Fuel compositions comprising tetramethylcyclohexane

ABSTRACT

A fuel composition comprises at least a tetramethylcyclohexane and a fuel component. The tetramethylcyclohexane can be used as a fuel component or as a fuel additive in the fuel composition. The fuel composition may further comprise a conventional fuel component selected from a petroleum-based fuel component such as diesel fuel, jet fuel, kerosene or gasoline; or a coal-based fuel component. Methods of making and using the fuel composition are also disclosed.

PRIOR RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 60/950,879, filed Jul. 20, 2007,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention encompasses, among other things, fuel compositionscomprising a tetramethylcyclohexane and methods of making and using thefuel compositions. In certain embodiments, the tetramethylcyclohexane isderived from a C₁₀ isoprenoid compound. In other embodiments, thetetramethylcyclohexane comprises a quaternary carbon. In furtherembodiments, the fuel compositions comprise a petroleum-based fuelcomponent.

BACKGROUND OF THE INVENTION

Biofuel is generally a fuel derived from biomass, i.e., recently livingorganisms or their metabolic byproducts, such as manure from animals.Biofuel is desirable because it is a renewable energy source, unlikeother natural resources such as petroleum, coal and nuclear fuels.Biofuel includes, inter alia, biologically produced alcohols, alkenesand derivatives thereof. Generally, such biologically produced biofuelcan be formed by the action of microbes and enzymes through fermentationof biomass. For example, methanol can be produced from fermentation ofwood or other organic materials or formed naturally in the anaerobicmetabolism of many varieties of bacteria. Similarly, ethanol can bemass-produced by fermentation of starch or sugar which can be found in awide variety of crops such as sugar cane, sugar beet and corn.Furthermore, various isoprenoid compounds can be prepared biologicallyfrom simple sugars using a host cell that has been modified to producethe desired isoprenoid compounds.

Recently, because of concerns over global warming, rising oil prices aswell as decreasing oil reserves and increasing political instability inoil producing countries around the world, there are renewed interestsfrom governments, industries and academics in biofuels, particularlybiologically produced alcohols for automobiles. However, alcohols suchas methanol, ethanol and propanol are volatile enough that they cancause engine vapor lock and evaporative emission problems. Furthermore,alcohols generally have a high affinity to water and therefore, theygenerally contain an undesirable amount of water that can causecorrosive problem to internal combustion engines that use them as fuels.As a result, there is a need for biofuels, such as non-alcoholicbiofuels, that have a low affinity toward water. Further, there is alsoa need for biofuels that can be made reliably and reproducibly for usein internal combustion engines such as gasoline engines.

SUMMARY OF THE INVENTION

Provided herein are fuel components, fuel compositions and methods ofmaking and using same. Embodiments of the fuel compositions disclosedherein are believed to satisfy the above-mentioned needs. In someembodiments, the fuel compositions comprise a tetramethylcyclohexane. Inother embodiments, the tetramethylcyclohexane can be used as the fuelcomposition itself, a major component of the fuel composition or a minorcomponent of the fuel composition. In still other embodiments, thetetramethylcyclohexane is an isoprenoid. In certain embodiments, thetetramethylcyclohexane is made by semi-chemical synthesis or a hybridmethod and involves a C₁₀ isoprenoid that is made by a bioengineeredmicroorganism. In some embodiments, the fuel compositions disclosedherein can be used as gasoline. In further embodiments, the fuelcompositions disclosed herein can be used to power internal combustionengines such as gasoline engines.

In one aspect, provided herein are fuel compositions comprising atetramethylcyclohexane in an amount of at least 5 wt. %, based on thetotal weight of the fuel composition, and a fuel component.

In certain embodiments, the fuel component is a petroleum-based fuelcomponent. In other embodiments, the petroleum-based fuel component isgasoline, jet fuel or kerosene. In further embodiments, the fuelcomponent is a coal-based fuel component.

In some embodiments, the fuel composition further comprises a fueladditive. In other embodiments, the fuel additive disclosed herein isselected from the group consisting of oxygenates, antioxidants, thermalstability improvers, cetane improvers, stabilizers, cold flow improvers,combustion improvers, anti-foams, anti-haze additives, corrosioninhibitors, lubricity improvers, icing inhibitors, injector cleanlinessadditives, smoke suppressants, drag reducing additives, metaldeactivators, dispersants, detergents, demulsifiers, dyes, markers,static dissipaters, biocides and combinations thereof.

In certain embodiments, the amount of the tetramethylcyclohexane is atmost about 30 wt. %, based on the total weight of the fuel composition.In other embodiments, the amount of the tetramethylcyclohexane is atmost about 20 wt. %, based on the total weight of the fuel composition.In further embodiments, the amount of the tetramethylcyclohexane is atmost about 10 wt. %, based on the total weight of the fuel composition.

In some embodiments, the fuel composition disclosed herein comprises

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;wherein the total amount of (a) and (b) is from about 1 wt. % to about99 wt. %, and the total amount of (c) and (d) is from 0.5 wt. % to about50 wt. %, based on the total weight of (a)-(d).

In certain embodiments, the fuel composition further comprises thefollowing compounds:

or at least one stereoisomer thereof;

and

or at least one stereoisomer thereof;wherein the amount of (e) is from 0 wt. % to about 50 wt. %, the amountof (f) is from about 0.1 wt. % to about 20 wt. %, and the amount of (g)is from about 0.1 wt. % to about 30 wt. %, based on the total weight of(a)-(g).

In some embodiments, the total amount of (a) and (b) in the fuelcomposition is from about 50 wt. % to about 99 wt. %, based on the totalweight of (a)-(g). In other embodiments, the total amount of (a) and (b)in the fuel composition is from about 80 wt. % to about 99 wt. %, basedon the total weight of (a)-(g).

In some embodiments, the total amount of (c) and (d) in the fuelcomposition is less than about 10 wt. %, based on the total weight of(a)-(g).

In another aspect, provided herein are methods of making a fuelcomposition comprising contacting pinene with hydrogen in the presenceof a hydrogenation catalyst to form at least a tetramethylcyclohexane.

In certain embodiments, the pinene is α-pinene, β-pinene or acombination thereof. In other embodiments, the hydrogenation catalystcomprises a ruthenium catalyst. In further embodiments, the rutheniumcatalyst is ruthenium on a support material. In still furtherembodiments, the support material is carbon.

In some embodiments, the method further comprises the step of mixing thetetramethylcyclohexane with a fuel component to make the fuelcomposition. In further embodiments, the fuel component is apetroleum-based fuel component. In still further embodiments, thepetroleum-based fuel component is gasoline, jet fuel or kerosene.

In another aspect, provided herein are vehicles comprising an internalcombustion engine; a fuel tank connected to the internal combustionengine; and a fuel composition in the fuel tank, wherein the fuelcomposition comprises at least 5 wt. % of a tetramethylcyclohexane,based on the total weight of the fuel composition, and a fuel component,and wherein the fuel composition is used to power the internalcombustion engine. In certain embodiments, the internal combustionengine is a gasoline engine.

In another aspect, provided herein are vehicles comprising an internalcombustion engine; a fuel tank connected to the internal combustionengine; and a fuel composition in the fuel tank, wherein the fuelcomposition is prepared by contacting pinene with hydrogen in thepresence of a hydrogenation catalyst, and wherein the fuel compositionis used to power the internal combustion engine.

In certain embodiments, the pinene is α-pinene, β-pinene or acombination thereof.

In some embodiments, the internal combustion engine is a gasolineengine.

In another aspect, provided herein are methods of making a fuelcomposition comprising

-   -   (a) contacting a cell capable of making pinene with a sugar        under conditions suitable for making pinene;    -   (b) converting the pinene to pinane;    -   (c) converting the pinane to at least a tetramethylcyclohexane;        and    -   (d) mixing the tetramethylcyclohexane with a fuel component to        make the fuel composition.

In certain embodiments, the pinene is converted to pinane by hydrogen inthe presence of a first hydrogenation catalyst. In other embodiments,the pinane is converted to tetramethylcyclohexane by hydrogen in thepresence of a second hydrogenation catalyst. In further embodiments, thefirst hydrogenation catalyst and the second hydrogenation catalyst arethe same. In still further embodiments, the first hydrogenation catalystand the second hydrogenation catalyst are different.

In another aspect, provided herein are methods of making an RBOBcomprising mixing a gasoline with a fuel composition comprising atetramethylcyclohexane having a quaternary carbon atom in thecyclohexane ring, wherein the RBOB has a Reid vapor pressure from about7.0 psi to about 15.0 psi, and wherein the amount of the amount of thetetramethylcyclohexane is from about 1 wt. % to about 50 wt. %, based onthe total weight of the fuel composition.

In some embodiments, the tetramethylcyclohexane is

or at least one stereoisomer thereof.

In other embodiments, the tetramethylcyclohexane is

or at least one stereoisomer thereof.

In another aspect, provided herein are fuel tanks containing a fuelcomposition, wherein the fuel composition comprises at least 5 wt. % ofa tetramethylcyclohexane, based on the total weight of the fuelcomposition, and a fuel component.

In further embodiments, the fuel tank is a vehicle fuel tank.

In another aspect, provided herein are fuel compositions comprising agasoline and at least one tetramethylcyclohexane having a quaternarycarbon atom in the cyclohexane ring, wherein the amount of thetetramethylcyclohexane is from about 1 wt. % to about 50 wt. %, based onthe total weight of the fuel composition.

In some embodiments, the fuel composition disclosed herein furthercomprises a fuel additive. In further embodiments, the fuel additive isselected from the group consisting of oxygenates, antioxidants, thermalstability improvers, cetane improvers, stabilizers, cold flow improvers,combustion improvers, anti-foams, anti-haze additives, corrosioninhibitors, lubricity improvers, icing inhibitors, injector cleanlinessadditives, smoke suppressants, drag reducing additives, metaldeactivators, dispersants, detergents, demulsifiers, dyes, markers,static dissipaters, biocides and combinations thereof.

In some embodiments, the tetramethylcyclohexane disclosed herein is

or at least one stereoisomer thereof.

In other embodiments, the tetramethylcyclohexane disclosed herein is

or at least one stereoisomer thereof.

In one aspect, provided herein are fuel compositions comprising apetroleum-based fuel component and at least one tetramethylcyclohexanehaving a quaternary carbon atom in the cyclohexane ring. In someembodiments, the amount of the tetramethylcyclohexane is at least about1 wt. %, based on the total weight of the fuel composition. In otherembodiments, the amount of the tetramethylcyclohexane is at least about5 wt. %, based on the total weight of the fuel composition.

In some embodiments, the tetramethylcyclohexane is

or at least one stereoisomer thereof.

In other embodiments, the tetramethylcyclohexane is

or at least one stereoisomer thereof.

In certain embodiments, the petroleum-based fuel component is gasoline,jet fuel, kerosene or a combination thereof. In other embodiments, theamount of the petroleum-based fuel component is at least about 40 wt. %and the amount of the tetramethylcyclohexane is from about 5 wt. % toabout 50 wt. %, based on the total weight of the fuel composition.

In some embodiments, the fuel composition disclosed herein comprises

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;wherein the total amount of (a) and (b) is from about 1 wt. % to about99 wt. %, and the total amount of (c) and (d) is from about 0.5 wt. % toabout 50 wt. %, based on the total weight of (a)-(d).

In certain embodiments, the fuel composition further comprises thefollowing compounds:

or at least one stereoisomer thereof;

and

or at least one stereoisomer thereof;wherein the amount of (e) is from 0 wt. % to about 50 wt. %, the amountof (f) is from about 0.1 wt. % to about 20 wt. %, and the amount of (g)is from about 0.1 wt. % to about 30 wt. %, based on the total weight of(a)-(g).

In some embodiments, the total amount of (a) and (b) in the fuelcomposition is from about 50 wt. % to about 99 wt. %, based on the totalweight of (a)-(g).

In another aspect, provided herein are methods of making a fuelcomposition comprising the steps of (a) contacting pinene with hydrogenin the presence of a hydrogenation catalyst to form at least onetetramethylcyclohexane having a quaternary carbon atom in thecyclohexane ring; and (b) mixing the tetramethylcyclohexane with a fuelcomponent to make the fuel composition. In some embodiments, the fuelcomponent is a petroleum-based fuel component.

In certain embodiments, the pinene is α-pinene, β-pinene or acombination thereof. In other embodiments, the hydrogenation catalystcomprises a ruthenium catalyst.

In another aspect, provided herein are vehicles comprising an internalcombustion engine; a fuel tank connected to the internal combustionengine; and a fuel composition in the fuel tank, wherein the fuelcomposition comprises at least one tetramethylcyclohexane having aquaternary carbon atom in the cyclohexane ring, and wherein the fuelcomposition is used to power the internal combustion engine. In certainembodiments, the internal combustion engine is a gasoline engine.

In certain embodiments, the fuel composition disclosed herein has a Reidvapor pressure from about 7.0 psi to about 15.0 psi. In otherembodiments, the fuel composition disclosed herein is an RBOB or aCARBOB. In further embodiments, the fuel composition disclosed hereinmeets the specifications of an RBOB or a CARBOB.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the mevalonate (“MEV”) pathwayfor the production of isopentenyl diphosphate (“IPP”).

FIG. 2 is a schematic representation of the DXP pathway for theproduction of IPP and dimethylallyl pyrophosphate (“DMAPP”). Dxs is1-deoxy-D-xylulose-5-phosphate synthase; Dxr is1-deoxy-D-xylulose-5-phosphate reductoisomerase (also known as IspC);IspD is 4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspE is4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspF is2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG is1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG); and ispHis isopentenyl/dimethylallyl diphosphate synthase.

FIG. 3 is a schematic representation of the conversion of one moleculeof IPP and one molecule of DMAPP to geranyl diphosphate (“GPP”). Anenzyme known to catalyze this step is, for example, geranyl diphosphatesynthase.

FIG. 4 is a schematic representation of plasmid maps of expressionplasmids pTrc99A-APS, pTrc99A-GTS, pTrc99A-TS, pTrc99A-BPS, pTrc99A-SSand pTrc99A-LMS.

FIGS. 5A and 5B show the distillation profiles of AMG-500 and variousblends of AMG-500 in CARBOB respectively.

DEFINITIONS

“Bioengineered compound” refers to a compound made by a host cell,including any archae, bacterial, or eukaryotic cells or microorganism.

“Biofuel” refers to any fuel that is derived from a biomass, i.e.,recently living organisms or their metabolic byproducts, such as manurefrom cows. It is a renewable energy source, unlike other naturalresources such as petroleum, coal and nuclear fuels.

“Bioengineered fuel” refers to a fuel made at least in part by a hostcell, including any archae, bacterial, or eukaryotic cells ormicroorganism.

“Fuel” refers to one or more hydrocarbons, one or more alcohols, one ormore fatty esters or a mixture thereof. Preferably, liquid hydrocarbonsare used. Fuel can be used to power internal combustion engines such asreciprocating engines (e.g., gasoline engines and diesel engines),Wankel engines, jet engines, some rocket engines, missile engines andgas turbine engines. In some embodiments, fuel typically comprises amixture of hydrocarbons such as alkanes, cycloalkanes and aromatichydrocarbons. In other embodiments, fuel comprises one or more of thesubstituted cycloalkanes disclosed herein.

“Fuel additive” refers to chemical components added to fuels to alterthe properties of the fuel, e.g., to improve engine performance, fuelhandling, fuel stability, or for contaminant control. Types of additivesinclude, but are not limited to, antioxidants, thermal stabilityimprovers, cetane improvers, stabilizers, cold flow improvers,combustion improvers, anti-foams, anti-haze additives, corrosioninhibitors, lubricity improvers, icing inhibitors, injector cleanlinessadditives, smoke suppressants, drag reducing additives, metaldeactivators, dispersants, detergents, demulsifiers, dyes, markers,static dissipaters, biocides and combinations thereof. The term“conventional additives” refers to fuel additives known to the skilledartisan, such as those described above, that are not the substitutedcycloalkanes disclosed herein.

“Fuel composition” refers to a fuel that comprises at least two fuelcomponents.

“Fuel component” refers to any compound or a mixture of compounds thatare used to formulate a fuel composition. There can be “major fuelcomponents” and “minor fuel components.” A major fuel component ispresent in a fuel composition by at least 50% by volume; and a minorfuel component is present in a fuel composition by less than 50%. Fueladditives are minor fuel components. The tetramethylcyclohexanesdisclosed herein can be major fuel components or minor fuel components,by themselves or in a mixture with other fuel components.

“Isoprenoid” and “isoprenoid compound” are used interchangeably hereinand refer to a compound capable of being derived from IPP.

“Isoprenoid starting material” refers to an isoprenoid compound that iscapable of being made by a host cell.

“C₁₀ isoprenoid” or “C₁₀ isoprenoid compound” refers to an isoprenoidconsisting of 10 carbon atoms. In certain embodiments, the C₁₀isoprenoid is a tetramethylcyclohexane disclosed herein such astetramethylcyclohexanes having a quaternary carbon, e.g.,1,1,2,3-tetramethylcyclohexane and 1,1,2,5-tetramethylcyclohexane.

“Jet fuel” refers to a fuel suitable for use in a jet engine.

“Petroleum-based fuel” refers to a fuel that includes a fractionaldistillate of petroleum.

“RBOB,” or Reformulated Blendstock for Oxygenate Blending refers to anon-oxygenated gasoline suitable for blending with an oxygenate, e.g.,ethanol. In certain embodiments, an RBOB meets the requirements of theU.S. Environmental Protection Agency under Section 211(k) of the CleanAir Act. “CARBOB” refers to an RBOB suitable for use in California asregulated by the California Air Resources Board. “AZRBOB” or ArizonaBlendstock for Oxygenate Blending refers to a non-oxygenated gasolinesuitable for blending with an oxygenate for use in Arizona. “LVRBOB” orLas Vegas Blendstock for Oxygenate Blending refers to a non-oxygenatedgasoline suitable for blending with an oxygenate for use in Las Vegas.

“Reid Vapor Pressure,” or “RVP” of a fuel composition refers to theabsolute vapour pressure exerted by the fuel composition at 100° F. Ingeneral, the higher the RVP value, the more readily the fuel compositionwill evaporate. The vapor pressure of a fuel composition may be measuredaccording to any standard method acceptable by those of skill in theart. In certain embodiments, the Reid vapor pressure is measuredaccording to ASTM D323-06.

As used herein, a composition that is a “substantially pure” compound issubstantially free of one or more other compounds, i.e., the compositioncontains greater than 80 vol. %, greater than 90 vol. %, greater than 95vol. %, greater than 96 vol. %, greater than 97 vol. %, greater than 98vol. %, greater than 99 vol. %, greater than 99.5 vol. %, greater than99.6 vol. %, greater than 99.7 vol. %, greater than 99.8 vol. %, orgreater than 99.9 vol. % of the compound; or less than 20 vol. %, lessthan 10 vol. %, less than 5 vol. %, less than 3 vol. %, less than 1 vol.%, less than 0.5 vol. %, less than 0.1 vol. %, or less than 0.01 vol. %of the one or more other compounds, based on the total volume of thecomposition.

As used herein, a composition that is “substantially free” of a compoundmeans that the composition contains less than 20 vol. %, less than 10vol. %, less than 5 vol. %, less than 4 vol. %, less than 3 vol. %, lessthan 2 vol. %, less than 1 vol. %, less than 0.5 vol. %, less than 0.1vol. %, or less than 0.01 vol. % of the compound, based on the totalvolume of the composition.

“Tetramethylcyclohexane” refers to a cyclohexane substituted with fourmethyl groups. The term also includes compounds wherein thetetramethylcyclohexane is further substituted with one or moreadditional substituents. In certain embodiments, thetetramethylcyclohexane comprises a quaternary carbon. In someembodiments, the tetramethylcyclohexane having a quaternary carbonincludes 1,1,2,3-tetramethylcyclohexane and1,1,2,5-tetramethylcyclohexane.

“Quaternary carbon” refers to a carbon atom bonded to four other carbonatoms with single bonds.

In the following description, all numbers disclosed herein areapproximate values, regardless whether the word “about” or “approximate”is used in connection therewith. They may vary by 1 percent, 2 percent,5 percent, or, sometimes, 10 to 20 percent. Whenever a numerical rangewith a lower limit, R^(L), and an upper limit, R^(U), is disclosed, anynumber falling within the range is specifically disclosed. Inparticular, the following numbers within the range are specificallydisclosed: R=R^(L)+k*(R^(U)−R^(L)), wherein k is a variable ranging from1 percent to 100 percent with a 1 percent increment, i.e., k is 1percent, 2 percent, 3 percent, 4 percent, 5 percent, . . . , 50 percent,51 percent, 52 percent, . . . , 95 percent, 96 percent, 97 percent, 98percent, 99 percent, or 100 percent. Moreover, any numerical rangedefined by two R numbers as defined in the above is also specificallydisclosed.

In addition, certain compounds, as described herein may have one or moredouble bonds that can exist as either the Z or E isomer, unlessotherwise indicated. The invention additionally encompasses thecompounds as individual isomers substantially free of other isomers andalternatively, as mixtures of various isomers, e.g., racemic mixtures ofstereoisomers.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Provided herein are fuel components, fuel compositions and methods ofmaking and using same.

Fuel Compositions

In one aspect, provided herein are fuel compositions comprising:

-   -   (a) a tetramethylcyclohexane in an amount of at least 0.5%,        based on the total amount of the fuel composition; and    -   (b) a fuel component.

The amount of the tetramethylcyclohexane in the fuel composition can befrom 0.5% to about 99%, from 0.5% to about 98%, from about 1% to about90%, from about 1% to about 75%, from about 1% to about 50%, from about1% to about 40%, from about 1% to about 30%, from about 5% to about 75%,from about 5% to about 60%, from about 5% to about 50%, from about 5% toabout 40%, or from about 5% to about 30%, based on the total amount ofthe fuel composition. In certain embodiments, the amount of thetetramethylcyclohexane is at least about 1%, at least about 2%, at leastabout 3%, at least about 4%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90% or at least about 95%, based on thetotal amount of the fuel composition. In other embodiments, the amountof the tetramethylcyclohexane is at most about 1%, at most about 2%, atmost about 3%, at most about 4%, at most about 5%, at most about 10%, atmost about 15%, at most about 20%, at most about 25%, at most about 30%,at most about 35%, at most about 40%, at most about 45%, at most about50%, at most about 55%, at most about 60%, at most about 65%, at mostabout 70%, at most about 75%, at most about 80%, at most about 85%, atmost about 90% or at most about 95%, based on the total amount of thefuel composition. In some embodiments, the amount is in wt. % based onthe total weight of the fuel composition. In other embodiments, theamount is in vol. % based on the total volume of the fuel composition.

In some embodiments, the total amount of the tetramethylcyclohexane inthe fuel compositions is from about 1% to about 99% by weight or volume,based on the total weight or volume of the fuel composition. In furtherembodiments, the total amount of the tetramethylcyclohexane is fromabout 25% to about 98% by weight or volume, based on the total weight orvolume of the fuel composition. In further embodiments, the total amountof the tetramethylcyclohexane is from about 50% to about 95% by weightor volume, based on the total weight or volume of the fuel composition.

In some embodiments, the tetramethylcyclohexane is or comprises atetramethylcyclohexane having at least one quaternary carbon atom in thecyclohexane ring. Some non-limiting examples of suitabletetramethylcyclohexanes having at least one quaternary carbon atominclude Compounds (1)-(9) as shown below and their stereoisomers:

In some embodiments, the tetramethylcyclohexane is or comprises1,1,2,3-tetramethylcyclohexane having formula (1):

or one or more stereoisomers thereof.

In other embodiments, the 1,1,2,3-tetramethylcyclohexane is or comprisesone or more of the following compounds:

In some embodiments, the tetramethylcyclohexane is or comprises1,1,2,5-tetramethylcyclohexane having formula (2)

or one or more stereoisomers thereof.

In other embodiments, the 1,1,2,5-tetramethylcyclohexane is or comprisesone or more of the following compounds:

In some embodiments, the tetramethylcyclohexane is or comprises both

or stereoisomers thereof.

In other embodiments, the tetramethylcyclohexane is or comprises one ormore of the following compounds:

In some embodiments, a fuel component is a petroleum-based fuelcomponent. The amount of the petroleum-based fuel component in the fuelcomposition disclosed herein may be from 0.1% to 99%, from 1% to 95%,from 2% to 90%, from 3% to 85%, from 5% to 80%, from 5% to 70%, from 5%to 60%, or from 5% to 50%, based on the total amount of the fuelcomposition. In certain embodiments, the amount of the petroleum-basedfuel component is less than 95%, less than 90%, less than 85%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, lessthan 50%, less than 45%, less than 40%, less than 35%, less than 30%,less than 25%, less than 20%, less than 15%, less than 10%, less than5%, less than 4%, less than 3%, less than 2%, less than 1% or less than0.5%, based on the total amount of the fuel composition. In otherembodiments, the amount of the petroleum-based fuel component is atleast 95%, at least 90%, at least 85%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55%, at least 50%, at least 45%, atleast 40%, at least 35%, at least 30%, at least 25%, at least 20%, atleast 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least2%, at least 1% or at least 0.5%, based on the total amount of the fuelcomposition. In some embodiments, the amount is in wt. % based on thetotal weight of the fuel composition. In other embodiments, the amountis in vol. % based on the total volume of the fuel composition.

In some embodiments, the petroleum-based fuel component is gasoline. Incertain embodiments, the gasoline meets one or more of the nine gasolineproperties as specified in ASTM D 4814 for gasoline, which isincorporated herein by reference. In general, conventional gasoline is amixture of hydrocarbons whose boiling point is below about 200° C.,obtained in the fractional distillation of petroleum. The hydrocarbonconstituents in the boiling range of gasoline are generally thosehydrocarbons having 4 to 12 carbon atoms. In general, gasoline can varywidely in composition; even gasolines with the same octane number may bequite different in composition.

In some embodiments, the fuel composition is an RBOB or meets thespecifications of an RBOB. In other embodiments, the fuel composition isa CARBOB or meets the specifications of a CARBOB. In furtherembodiments, the fuel composition is an AZRBOB or meets thespecifications of AZRBOB. In further embodiments, the fuel compositionis an LVRBOB or meets the specifications of LVRBOB. In certainembodiments, provided herein are fuel compositions comprising an RBOBfuel composition, as described herein, and an oxygenate. In certainembodiments, provided herein are fuel compositions comprising an RBOBfuel composition, as described herein, and ethanol. In certainembodiments, provided herein are fuel compositions comprising a CARBOBfuel composition, as described herein, and an oxygenate. In certainembodiments, provided herein are fuel compositions comprising a CARBOBfuel composition, as described herein, and ethanol. In certainembodiments, provided herein are fuel compositions comprising an AZRBOBfuel composition, as described herein, and an oxygenate. In certainembodiments, provided herein are fuel compositions comprising an AZRBOBfuel composition, as described herein, and ethanol. In certainembodiments, provided herein are fuel compositions comprising an LVRBOBfuel composition, as described herein, and an oxygenate. In certainembodiments, provided herein are fuel compositions comprising an LVRBOBfuel composition, as described herein, and ethanol.

In certain embodiments, provided herein are fuel compositions that meetfederal or regional seasonal requirements or specifications for Reidvapor pressure (RVP). Certain fuel compositions or components disclosedherein can have a low RVP. As such, they can be blended with other fuelcomponents, e.g., gasoline fuel components, with a higher RVP to adjustor lower the RVP of the resulting fuel composition. Accordingly,provided herein are methods of adjusting the RVP of a fuel componentcomprising the step of adding to the fuel component a fuel compositiondisclosed herein, for example, a fuel composition comprising atetramethylcyclohexane as disclosed herein.

In certain embodiments, provided herein are fuel compositions having anRVP from about 7.0 pounds per square inch (psi) to about 15.0 psi. Insome embodiments, provided herein are fuel compositions having an RVPfrom about 8.0 to about 10.0 psi. In certain embodiments, providedherein are fuel compositions having an RVP of about 5.78 psi. In someembodiments, provided herein are fuel compositions having an RVP ofabout 6.8 psi. In certain embodiments, provided herein are fuelcompositions having an RVP of about 7.0 psi. In some embodiments,provided herein are fuel compositions having an RVP of about 7.1 psi. Incertain embodiments, provided herein are fuel compositions having an RVPof about 7.8 psi. In some embodiments, provided herein are fuelcompositions having an RVP of about 8.0 psi. In certain embodiments,provided herein are fuel compositions having an RVP of about 8.5 psi. Insome embodiments, provided herein are fuel compositions having an RVP ofabout 9.0 psi. In certain embodiments, provided herein are fuelcompositions having an RVP of about 10.0 psi. In some embodiments,provided herein are fuel compositions having an RVP of about 11.0 psi.In certain embodiments, provided herein are fuel compositions having anRVP of about 11.5 psi. In some embodiments, provided herein are fuelcompositions having an RVP of about 12.5 psi. In certain embodiments,provided herein are fuel compositions having an RVP of about 13.5 psi.In some embodiments, provided herein are fuel compositions having an RVPof about 14.0 psi. In certain embodiments, provided herein are fuelcompositions having an RVP of about 15.0 psi.

In other embodiments, the petroleum-based fuel component is kerosene.Conventional kerosene in general is a mixture of hydrocarbons, having aboiling point from 285° F. to 610° F. (from 140° C. to 320° C.).

In still other embodiments, the petroleum-based fuel component is jetfuel. Any jet fuel known to skilled artisans can be used herein. TheAmerican Society for Testing and Materials (“ASTM”) and the UnitedKingdom Ministry of Defense (“MOD”) have taken the lead roles in settingand maintaining specification for civilian aviation turbine fuel or jetfuel. The respective specifications issued by these two organizationsare very similar but not identical. Many other countries issue their ownnational specifications for jet fuel, but they can be very nearly orcompletely identical to either the ASTM or MOD specification. ASTM D1655 is the Standard Specification for Aviation Turbine Fuels andincludes specifications for Jet A, Jet A-1 and Jet B fuels. DefenceStandard 91-91 is the MOD specification for Jet A-1.

The most common jet fuel is a kerosene/paraffin oil-based fuelclassified as Jet A-1, which is produced to an internationallystandardized set of specifications. In the United States only, a versionof Jet A-1 known as Jet A is also used. Another jet fuel that iscommonly used in civilian aviation is called Jet B. Jet B is a lighterfuel in the naptha-kerosene region that is used for its enhancedcold-weather performance. Jet A, Jet A-1 and Jet B are specified in ASTMSpecification D. 1655-68. Alternatively, jet fuels are classified bymilitaries around the world with a different system of JP numbers. Someare almost identical to their civilian counterparts and differ only bythe amounts of a few additives. For example, Jet A-1 is similar to JP-8and Jet B is similar to JP-4. Alternatively, jet fuels can also beclassified as kerosene or naphtha-type. Some non-limiting examples ofkerosene-type jet fuels include Jet A, Jet A1, JP-5 and JP-8. Somenon-limiting examples of naphtha-type jets fuels include Jet B and JP-4.

Jet A is used in the United States while most of the rest of the worlduses Jet A-1. Jet A is similar to Jet-A1, except for its higher freezingpoint of −40° C. An important difference between Jet A and Jet A-1 isthe maximum freezing point. Jet A-1 has a lower maximum freezingtemperature of −47° C. while Jet A has a maximum freezing temperature of−40° C. Like Jet A-1, Jet A has a fairly high flash point of minimum 38°C., with an autoignition temperature of 210° C.

In certain embodiments, a fuel component is a fuel additive. In someembodiments, the fuel additive is from about 0.1% to less than 50% byweight or volume, based on the total weight or volume of the fuelcomposition. In further embodiments, the fuel additive is selected fromthe group consisting of oxygenates, antioxidants, thermal stabilityimprovers, cetane improvers, stabilizers, cold flow improvers,combustion improvers, anti-foams, anti-haze additives, corrosioninhibitors, lubricity improvers, icing inhibitors, injector cleanlinessadditives, smoke suppressants, drag reducing additives, metaldeactivators, dispersants, detergents, demulsifiers, dyes, markers,static dissipaters, biocides and combinations thereof.

The amount of a fuel additive in the fuel composition disclosed hereinmay be from about 0.1% to less than about 45%, from about 0.2% to about40%, from about 0.3% to about 30%, from about 0.4% to about 20%, fromabout 0.5% to about 15% or from about 0.5% to about 10%, based on thetotal amount of the fuel composition. In certain embodiments, the amountof a fuel additive is less than about 50%, less than about 45%, lessthan about 40%, less than about 35%, less than about 30%, less thanabout 25%, less than about 20%, less than about 15%, less than about10%, less than about 5%, less than about 4%, less than about 3%, lessthan about 2%, less than about 1% or less than about 0.5%, based on thetotal amount of the fuel composition. In some embodiments, the amount isin wt. % based on the total weight of the fuel composition. In otherembodiments, the amount is in vol. % based on the total volume of thefuel composition.

Some conventional fuel additives have been described in “Gasoline:Additives, Emissions, and Performance” by Society of AutomotiveEngineers, SAE International, 1995 (ISBN: 1560916451), which isincorporated herein by reference. Further, the following U.S. patentsdisclose various fuel additives that can be employed in embodiments ofthe invention as additives: U.S. Pat. Nos. 6,054,420; 6,051,039;5,997,593; 5,997,592; 5,993,498; 5,968,211; 5,958,089; 5,931,977;5,891,203; 5,882,364; 5,880,075; 5,880,072; 5,855,629; 5,853,436;5,743,922; 5,630,852; 5,529,706; 5,505,867; 5,492,544; 5,490,864;5,484,462; 5,321,172; and 5,284,492. The disclosures of all of thepreceding U.S. patents are incorporated by reference herein in theirentirety.

Illustrative examples of fuel additives are described in greater detailbelow. Oxygenates, which increase the weight % of oxygen in the fuelcomposition, are one example. Generally, oxygenates are combustibleliquids comprising carbon, hydrogen and oxygen that can be categorizedinto two classes of organic compounds, i.e., alcohols and ethers. Somenon-limiting examples of suitable oxygenates include ethanol, methyltertiary-butyl ether (MTBE), tertiary-amyl methyl ether (TAME), andethyl tertiary-butyl ether (ETBE).

Lubricity improvers are another example. Typically, the concentration ofthe lubricity improver in the fuel falls in the range of from 1 to50,000 ppm, preferably about 10 to 20,000 ppm, and more preferably from25 to 10,000 ppm. Some non-limiting examples of lubricity improverinclude esters of fatty acids.

Stabilizers improve the storage stability of the fuel composition. Somenon-limiting examples of stabilizers include tertiary alkyl primaryamines. The stabilizer may be present in the fuel composition at aconcentration of about 0.001 to about 2 wt %, based on the total weightof the fuel composition, and in one embodiment from about 0.01 to about1% by weight.

Combustion improvers increase the mass burning rate of the fuelcomposition. Some non-limiting examples of combustion improvers includeferrocene(dicyclopentadienyl iron), iron-based combustion improvers(e.g., TURBOTECT™ ER-18 from Turbotect (USA) Inc., Tomball, Tex.),barium-based combustion improvers, cerium-based combustion improvers,and iron and magnesium-based combustion improvers (e.g., TURBOTECT™ 703from Turbotect (USA) Inc., Tomball, Tex.). The combustion improver maybe present in the fuel composition at a concentration of about 0.001 toabout 1 wt %, based on the total weight of the fuel composition, and inone embodiment from about 0.01 to about 1% by weight.

Antioxidants prevent the formation of gum depositions on fuel systemcomponents caused by oxidation of fuels in storage and/or inhibit theformation of peroxide compounds in certain fuel compositions can be usedherein. The antioxidant may be present in the fuel composition at aconcentration of about 0.001 to about 5 wt %, based on the total weightof the fuel composition, and in one embodiment from about 0.01 to about1% by weight.

Static dissipaters reduce the effects of static electricity generated bymovement of fuel through high flow-rate fuel transfer systems. Thestatic dissipater may be present in the fuel composition at aconcentration of about 0.001 to about 5 wt %, based on the total weightof the fuel composition, and in one embodiment from about 0.01 to about1% by weight.

Corrosion inhibitors protect ferrous metals in fuel handling systemssuch as pipelines, and fuel storage tanks, from corrosion. Incircumstances where additional lubricity is desired, corrosioninhibitors that also improve the lubricating properties of thecomposition can be used. The corrosion inhibitor may be present in thefuel composition at a concentration of about 0.001 to about 5 wt %,based on the total weight of the fuel composition, and in one embodimentfrom about 0.01 to about 1% by weight.

Fuel system icing inhibitors (also referred to as anti-icing additive)reduce the freezing point of water precipitated from jet fuels due tocooling at high altitudes and prevent the formation of ice crystalswhich restrict the flow of fuel to the engine. Certain fuel system icinginhibitors can also act as a biocide. The fuel system icing inhibitormay be present in the fuel composition at a concentration of about 0.001to about 5 wt %, based on the total weight of the fuel composition, andin one embodiment from about 0.01 to about 1% by weight.

Biocides are used to combat microbial growth in the fuel composition.The biocide may be present in the fuel composition at a concentration ofabout 0.001 to about 5 wt %, based on the total weight of the fuelcomposition, and in one embodiment from about 0.01 to about 1% byweight.

Metal deactivators suppress the catalytic effect of some metals,particularly copper, have on fuel oxidation. The metal deactivator maybe present in the fuel composition at a concentration of about 0.001 toabout 5 wt %, based on the total weight of the fuel composition, and inone embodiment from about 0.01 to about 1% by weight.

Thermal stability improvers are use to inhibit deposit formation in thehigh temperature areas of the aircraft fuel system. The thermalstability improver may be present in the fuel composition at aconcentration of about 0.001 to about 5 wt %, based on the total weightof the fuel composition, and in one embodiment from about 0.01 to about1% by weight

In some embodiments, the fuel compositions disclosed herein furthercomprise an aromatic compound. In some embodiments, the aromaticcompound is or comprises an isoprenoid compound. In other embodiments,the aromatic compound is or comprises a C₁₀ isoprenoid compound.

In some embodiments, the aromatic compound is or comprises

In other embodiments, the aromatic compound is or comprises

In certain embodiments, the aromatic compound is or comprises

In some embodiments, the total amount of aromatic compounds in the fuelcompositions is from about 1% to about 50% by weight or volume, based onthe total weight or volume of the fuel composition. In otherembodiments, the total amount of aromatic compounds in the fuelcompositions is from about 10% to about 35% by weight or volume, basedon the total weight or volume of the fuel compositions. In furtherembodiments, the total amount of aromatic compounds in the fuelcompositions is from about 10% to about 25% by weight or volume, basedon the total weight or volume of the fuel compositions. In still furtherembodiments, the total amount of aromatic compounds in the fuelcompositions is less than about 25% by weight or volume, based on thetotal weight or volume of the fuel compositions.

In some embodiments, the fuel compositions disclosed herein furthercomprise a methylisopropylcyclohexane. In certain embodiments, themethylisopropylcyclohexane is:

In other embodiments, the methylisopropylcyclohexane is or comprises oneor more of the following compounds:

In certain other embodiments, the methylisopropylcyclohexane is orcomprises

In other embodiments, the methylisopropylcyclohexane is or comprises oneor more of the following compounds:

In another aspect, provided herein are fuel compositions comprising:

or at least a stereoisomer thereof;

or at least a stereoisomer thereof;

or at least a stereoisomer thereof; and

or at least a stereoisomer thereof,wherein (a), (b), (c), and (d) are each present in an amount of at least0.5% by volume or weight, based on the total volume or weight of thefuel composition. In other embodiments, (a), (b), (c), and (d) are eachpresent in an amount of at least 1% by volume or weight, based on thetotal volume or weight of the fuel composition.

In some embodiments, the fuel compositions disclosed herein furthercomprise an aromatic compound. In other embodiments, the aromaticcompound is a C₁₀ isoprenoid selected from the group consisting of:

and combinations thereof.

In certain embodiments, the fuel compositions disclosed herein furthercomprise the following compounds:

or at least one stereoisomer thereof;

and

or at least one stereoisomer thereof.

In some embodiments, the fuel composition disclosed herein comprises

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

and

or at least one stereoisomer thereof.

In some embodiments, the amount of (a), (b), (c), (d), (e), (f) or (g)in wt. % or vol. % is from 0% to about 99%, from 0% to about 50%, from0% to about 40%, from 0% to about 30%, from 0% to about 20%, from 0% toabout 10%, from 0% to about 5%, from about 0.1% to about 99%, from about0.1% to about 50%, from about 0.1% to about 40%, from about 0.1% toabout 30%, from about 0.1% to about 20%, from about 0.1% to about 10%,from about 0.1% to about 5%, from about 0.5% to about 99%, from about0.5% to about 50%, from about 0.5% to about 40%, from about 0.5% toabout 30%, from about 0.5% to about 20%, from about 0.5% to about 10%,from about 0.5% to about 5%, from about 1% to about 99%, from about 1%to about 50%, from about 1% to about 40%, from about 1% to about 30%,from about 1% to about 20%, from about 1% to about 10%, from about 1% toabout 5%, from about 50% to about 99%, from about 60% to about 99%, fromabout 70% to about 99%, from about 80% to about 99%, or from about 90%to about 99%, based on the total weight or volume of (a)-(d) or (a)-(g)or the fuel composition.

In other embodiments, the amount of (a), (b), (c), (d), (e), (f) or (g)in wt. % or vol. % is less than about 1%, less than about 3%, less thanabout 5%, less than about 10%, less than about 20%, less than about 30%,less than about 40%, less than about 50%, less than about 60%, less thanabout 70%, less than about 80%, less than about 85%, less than about90%, or less than about 95%, based on the total weight or volume of(a)-(d) or (a)-(g) or the fuel composition.

In further embodiments, the amount of (a), (b), (c), (d), (e), (f) or(g) in wt. % or vol. % is at least about 0.1%, at least about 0.3%, atleast about 0.5%, at least about 1%, at least about 3%, at least about5%, at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, based onthe total weight or volume of (a)-(d) or (a)-(g) or the fuelcomposition.

In some embodiments, the amount of (a) and (b) in wt. % or vol. % isfrom about 1% to about 99%, from about 10% to about 99%, from about 20%to about 99%, from about 30% to about 99%, from about 40% to about 99%,from about 50% to about 99%, from about 60% to about 99%, from about 70%to about 99%, from about 80% to about 99%, or from about 90% to about99%, based on the total weight or volume of (a)-(d) or (a)-(g) or thefuel composition. In other embodiments, the total amount of (a) and (b)in wt. % or vol. % is at least about 0.5%, at least about 1%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90%, based on the total weight or volume of(a)-(d) or (a)-(g) or the fuel composition.

In some embodiments, the amount of tetramethylcyclohexane in wt. % orvol. % is from about 1% to about 99%, from about 10% to about 99%, fromabout 20% to about 99%, from about 30% to about 99%, from about 40% toabout 99%, from about 50% to about 99%, from about 60% to about 99%,from about 70% to about 99%, from about 80% to about 99%, or from about90% to about 99%, based on the total weight or volume of (a)-(d) or(a)-(g) or the fuel composition. In other embodiments, the amount oftetramethylcyclohexane in wt. % or vol. % is at least about 0.5%, atleast about 1%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, or at least about 95%, based on the totalweight or volume of the fuel composition.

In certain embodiments, the total amount of (c) and (d) in wt. % or vol.% is from 0% to about 50%, from 0.1% to about 50%, from about 0.1% toabout 40%, from about 0.1% to about 30%, from about 0.1% to about 20%,from about 0.1 % to about 10%, from about 0.1% to about 5%, or fromabout 0% to about 5%, from 0.5% to about 50%, from about 0.5% to about40%, from about 0.5% to about 30%, from about 0.5% to about 20%, fromabout 0.5% to about 10%, from about 0.5% to about 5%, or from about 0%to about 5%, based on the total weight or volume of (a)-(d) or (a)-(g)or the fuel composition. In other embodiments, the total amount of (c)and (d) in wt. % or vol. % is less than about 50%, less than about 40%,less than about 30%, less than about 20%, less than about 10%, less thanabout 5%, less than about 3%, or less than about 1%, based on the totalweight or volume of (a)-(d) or (a)-(g) or the fuel composition.

In some embodiments, the amounts disclosed herein are in wt. % based onthe total weight of (a)-(d). In other embodiments, the amounts disclosedherein are in wt. % based on the total weight of (a)-(g). In furtherembodiments, the amounts disclosed herein are in wt. % based on thetotal weight of the fuel composition. In certain embodiments, theamounts disclosed herein are in vol. % based on the total volume of(a)-(d). In other embodiments, the amounts disclosed herein are in vol.% based on the total volume of (a)-(g). In further embodiments, theamounts disclosed herein are in vol. % based on the total volume of thefuel composition.

In some embodiments, the fuel compositions further comprise a fuelcomponent. In still other embodiments, the fuel component is orcomprises a petroleum-based fuel component. In still other embodiments,the fuel component is or comprises a fuel additive.

Methods for Making Fuel Compositions

In another aspect, provided herein are methods of making a fuelcomposition comprising the steps of:

-   -   contacting pinene with hydrogen in the presence of a catalyst to        form a tetramethylcyclohexane; and    -   mixing the tetramethylcyclohexane with a fuel component to make        the fuel composition.

In some embodiments, the methods comprise the step of contacting pinenewith hydrogen in the presence of a hydrogenation catalyst to form atleast one tetramethylcyclohexane having a quaternary carbon atom in thecyclohexane ring.

In some embodiments, the pinene is α-pinene:

In other embodiments, the pinene is β-pinene:

In some embodiments, the pinene is α-pinene, β-pinene or a combinationthereof. In other embodiments, the pinene is made by host cells.

In certain embodiments, the hydrogenation of pinene totetramethylcyclohexane is a one-pot reaction or synthesis. One-potreaction or synthesis refers to a chemical process whereby a reactant issubjected to a single reaction or successive chemical reactions in justone reactor.

In other embodiments, the hydrogenation of pinene totetramethylcyclohexane comprises two steps. In the first step, pinene ishydrogenated to pinane. Subsequently, pinane is further hydrogenated totetramethylcyclohexane.

Whether hydrogenation is one pot reaction or comprises two or moredifferent reactions, the hydrogenation typically occurs by reacting oneor more of the reactants with hydrogen in the presence of ahydrogenation catalyst such as Pd, Pd/C, Pt, PtO₂, Rh, Ru(PPh₃)₂Cl₂,nickel, Raney nickel and combinations thereof. In some embodiments, thecatalyst is a ruthenium catalyst such as Rh or Ru(PPh₃)₂Cl₂. In otherembodiments, the catalyst is a rhodium catalyst such as Rh. In certainembodiments, the catalyst is a palladium catalyst such as Pd or Pd/C. Infurther embodiments, the catalyst is a platinum catalyst such as Pt orPtO₂. In further embodiments, the catalyst is a nickel catalyst such asnickel or Raney nickel.

The hydrogenation catalyst can have a surface area between about 25 m²/gand about 300 m²/g. In some embodiments, the surface area of thehydrogenation catalyst is between about 50 m²/g and about 250 m²/g. Inother embodiments, the surface area of the hydrogenation catalyst isbetween about 70 m²/g and about 250 m²/g. In further embodiments, thesurface area of the hydrogenation catalyst is between about 50 m²/g andabout 200 m²/g. In certain embodiments, the surface area of thehydrogenation catalyst is between about 70 m²/g and about 150 m²/g. Thehydrogenation catalyst can have an average particle size ranging fromabout 5 to about 300 microns, from about 20 to about 250 microns, fromabout 20 to about 200 microns, from about 20 to about 150 microns, fromabout 20 to about 120 microns, from about 30 to about 100 microns, orfrom about 30 to about 90 microns.

The hydrogenation catalyst can be distributed, coated, deposited orsupported on a support material. Any support material which is known inthe art to be suitable as a support for hydrogenation catalyst can beused herein. Non-limiting examples of suitable support materials includecarbon such as activated carbon, alumina such as activated alumina ormicrogel alumina, silica, silica-alumina, alumina silicates, magnesia,kieselguhr, fuller's earth, clays, porous rare earth halides andoxylalides, and combinations thereof.

The support material can be in the form of particles have a surface areabetween about 5 m²/g and about 450 m²/g. In some embodiments, thesurface area of the support material is between about 10 m²/g and about400 m²/g. In further embodiments, the surface area of the supportmaterial is between about 15 m²/g and about 350 m²/g. In certainembodiments, the surface area of the support material is between about20 m²/g and about 300 m²/g. The support material can have an averageparticle size ranging from about 5 to about 300 microns, from about 10to about 250 microns, from about 15 to about 200 microns, from about 20to about 150 microns, or from about 20 to about 120 microns.

The surface area of the hydrogenation catalyst or the support materialcan be determined by the BET (Brunauer-Emmet-Teller) method of measuringsurface area, as described by S. Brunauer, P. H. Emmett, and E. Teller,Journal of the American Chemical Society, 60, 309 (1938), which isincorporated herein by reference. The average particle sizes of thehydrogenation catalyst or the support material can be measured by anyparticle size measurement method known to a skilled artisan. Forexample, the average particle size of the support material can beobtained by ASTM D4460-00 or any similar method known to a personskilled in the art.

Generally, after completion of the hydrogenation, the reaction mixturecan be washed, concentrated, and dried to yield the desired hydrogenatedproduct. Alternatively, any reducing agent that can reduce a C═C bond toa C—C bond can also be used. An illustrative example of such a reducingagent is hydrazine in the presence of a catalyst, such as5-ethyl-3-methyllumiflavinium perchlorate, under an oxygen atmosphere.The reduction reaction with hydrazine is disclosed in Imada et al., J.Am. Chem. Soc., 127, 14544-14545 (2005), which is incorporated herein byreference.

In other embodiments, the hydrogenation reaction is carried out in thepresence of an asymmetric hydrogenation catalyst such as rhodium-chiraldiphosphine complex to form stereospecific hydrogenated productssubstantially free of other stereoisomers. A non-limiting example of theasymmetric hydrogenation catalyst includes the rhodium-DIPAMP catalyst.The rhodium-DIPAMP catalyst and other asymmetric hydrogenation catalystsare disclosed in Vineyard et al., J. Am. Chem. Soc. 1977, 99, (18),5946; Ryoji Noyori, “Asymmetric Catalysis In Organic Synthesis,” JohnWiley & Sons Inc., New York, Chapter 2, pp. 16-94 (1994); and Blaser etal., “Asymmetric Catalysis on Industrial Scale: Challenges, Approachesand Solutions,” Wiley-VCH, Weinheim, pp. 23-52 (2004), all of which areincorporated herein by reference in their entirety.

In some embodiments, the hydrogenation reaction occurs in two steps. Inthe first step, as shown in Scheme 1.

In other embodiments, α-pinene or β-pinene can be hydrogenated in thepresence of an asymmetric hydrogenation catalyst to form preferentiallyor substantially one of two possible stereoisomers of pinane, as shownbelow:

Once pinene is converted to pinane, a subsequent hydrogenation reactioncan convert pinane to one or more tetramethylcyclohexanes as shown inScheme 2.

In some embodiments, the same catalyst and reaction conditions for thefirst step is used in the second step. In other embodiments, a differentcatalyst is used for the second hydrogenation reaction. In still otherembodiments, the same catalyst is used but different reaction conditionsare applied. In further embodiments, the conversion of pinene to one ormore tetramethylcyclohexanes occurs in a one-pot reaction. In stillfurther embodiments, the conversion of pinene to one or moretetramethylcyclohexanes occurs in a single step reaction wherein pinaneis an intermediate of the single step reaction.

Depending on the temperature and pressure of the reaction of Scheme 2,the reaction may yield additional products such as:

Another additional product of the reaction of Scheme 2 may bedimethyloctane:

In certain embodiments, the pinane is trans-pinane (25). In otherembodiments, the pinane is cis-pinane (26). In still other embodiments,the pinane is a mixture of cis-pinane (26) and trans-pinane (25).

In another aspect, provided herein are methods of making a fuelcomposition from a sugar or a non-fermentable carbon source, comprisingthe steps of:

-   -   (a) contacting a cell capable of making pinene with the sugar        under conditions suitable for making pinene;    -   (b) converting the pinene to pinane;    -   (c) converting the pinane to at least a tetramethylcyclohexane;        and,    -   (d) mixing the tetramethylcyclohexane with a fuel component to        make the fuel composition.

The sugar can be any sugar known to those of skill in the art. Somenon-limiting examples of suitable monosaccharides include glucose,galactose, mannose, fructose, ribose and combinations thereof. Somenon-limiting examples of suitable disaccharides include sucrose,lactose, maltose, trehalose, cellobiose and combinations thereof. Incertain embodiments, the bioengineered fuel component can be obtainedfrom a polysaccharide. Some non-limiting examples of suitablepolysaccharides include starch, glycogen, cellulose, chitin andcombinations thereof.

The monosaccharides, disaccharides and polysaccharides suitable formaking the bioengineered tetramethylcyclohexane can be found in a widevariety of crops or sources. Some non-limiting examples of suitablecrops or sources include sugar cane, bagasse, miscanthus, sugar beet,sorghum, grain sorghum, switchgrass, barley, hemp, kenaf, potatoes,sweet potatoes, cassava, sunflower, fruit, molasses, whey or skim milk,corn, stover, grain, wheat, wood, paper, straw, cotton, many types ofcellulose waste, and other biomass. In certain embodiments, the suitablecrops or sources include sugar cane, sugar beet and corn.

A non-fermentable carbon source is a carbon source that cannot beconverted by the organism into ethanol. Some non-limiting examples ofsuitable non-fermentable carbon sources include acetate and glycerol.

Methods for Making Compounds

The compounds of the present invention can be made using any methodknown in the art including biologically, total chemical synthesis(without the use of biologically derived materials), and a hybrid methodwhere both biologically and chemical means are used.

Aromatic Isoprenoid Compounds

In certain embodiments, the inventive fuel compositions comprise anaromatic isoprenoid compound. Some illustrative examples of suitablearomatic isoprenoid compounds include:

In some embodiments, the aromatic isoprenoid compound is made byconverting an isoprenoid starting material into the correspondingaromatic compound by hydrogenation catalysts at a reaction temperaturebetween about 300° C. to about 350° C. Some illustrative examples ofsuitable hydrogenation catalysts include but are not limited toplatinum, palladium, and nickel. In general, milder conditions can beused if a hydrogen acceptor is present to remove hydrogen as it isformed.

In certain embodiments, the catalyst is platinum on activated alumina.In other embodiments, the catalyst is 5% platinum on activated alumina.In further embodiments, the catalyst loading is from about 1 gram perliter of substrate to about 50 grams per liter of substrate. In otherembodiments, the catalyst loading is less than about 25 grams per literof substrate. In other embodiments, the catalyst loading is less thanabout 10 grams per liter of substrate.

In other embodiments, the aromatic compound is or comprises both

In certain other embodiments, compounds 11 and 12 are made according toScheme 3.

In some embodiments, compounds 14 and 17 are derived from thehydrogenation reaction of pinene.

In other embodiments, the aromatic compound is or comprises

In certain other embodiments, compound 11 is made according to Scheme 4from limonene or γ-terpinene or terpinolene.

In other embodiments, the aromatic compound is or comprises

In certain other embodiments, compound 13 is made according to Scheme 5from sabinene.

The first step of Scheme 5 is a ring-opening reaction. Because of itsstrained bicyclic structure, isoprenoids like sabinane generally can beconverted by reductive ring-opening reactions to the corresponding lessstrained monocyclic alkanes. Any reductive ring-opening reagent that canreductively ring open a bicyclic alkane to the corresponding monocyclicalkane can be used herein. Some non-limiting examples of suitablereductive ring-opening reagents include hydrides such asdiisobutylaluminum hydride; hydrogen in the presence of a suitablecatalyst; and asymmetric reductive ring agents such as a mixture of anorganic acid, zinc powder and Ni(binap)Cl2 or Pd(binap)I2 as catalyst,where binap is 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl. Some ofthese asymmetric reductive ring agents are disclosed in Lautens, et al.,“Scope of the Nickel Catalyzed Asymmetric Reductive Ring OpeningReaction. Synthesis of Enantiomerically Enriched Cyclohexenols,”Tetrahedron, 54, 1107-1116 (1998); and Li et al., “Asymmetric ReductiveRing-Opening of Bicyclic Olefins Catalyzed by Palladium and NickelComplexes,” Org. Lett., 5(10), 1621-1624, 2003, both of which areincorporated herein by reference. Once sabinene is converted into itscorresponding monocyclic structure, it can be aromatized as describedpreviously.

In some embodiments, limonene, pinene, sabinene, and γ-terpinene areeach made from renewable sources, namely by the conversion of a carbonsource such as sugar to the desired isoprenoid starting material by hostcells.

Host Cell

A C₁₀ isoprenoid compound or starting material can be made by any methodknown in the art including biological methods, chemical syntheses, andhybrid methods. When the C₁₀ isoprenoid compound or starting material ismade biologically, one method is where a host cell that has beenmodified to produce the desired product. Like all isoprenoids, a C₁₀isoprenoid compound or starting material is made biochemically through acommon intermediate, isopentenyl diphosphate (“IPP”) or dimethylallylpyrophosphate (“DMAPP”).

Any suitable host cell may be used in the practice of the presentinvention. In one embodiment, the host cell is a genetically modifiedhost microorganism in which nucleic acid molecules have been inserted,deleted or modified (i.e., mutated; e.g., by insertion, deletion,substitution, and/or inversion of nucleotides), to either produce thedesired isoprenoid compound or starting material, or increased yields ofthe desired isoprenoid compound or starting material. In anotherembodiment, the host cell is capable of being grown in liquid growthmedium.

Illustrative examples of suitable host cells include any archae,bacterial, or eukaryotic cell. Examples of an archae cell include, butare not limited to those belong to the genera: Aeropyrum, Archaeglobus,Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus,and Thermoplasma. Illustrative examples of archae species include butare not limited to: Aeropyrum pernix, Archaeoglobus fulgidus,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Pyrococcus abyssi, Pyrococcus horikoshii, Thermoplasma acidophilum, andThermoplasma volcanium.

Examples of a bacterial cell include, but are not limited to thosebelonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena,Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium,Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia,Escherichia, Lactobacillus, Lactococcus, Mesorhizobium,Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter,Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun,Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, andZymomonas.

Illustrative examples of bacterial species include but are not limitedto: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacteriumammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii,Enterobacter sakazakii, Escherichia coli, Lactococcus lactis,Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii,Pseudomonas pudica, Rhodobacter caps ulatus, Rhodobacter sphaero ides,Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonellatyphimurium, Shigella dysenteriae, Shigellaflexneri, Shigella sonnei,Staphylococcus aureus, and the like.

In general, if a bacterial host cell is used, a non-pathogenic strain ispreferred. Illustrative examples of non-pathogenic species include butare not limited to: Bacillus subtilis, Escherichia coli, Lactibacillusacidophilus, Lactobacillus helveticus, Pseudomonas aeruginosa,Pseudomonas mevalonii, Pseudomonas pudita, Rhodobacter sphaeroides,Rodobacter capsulatus, Rhodospirillum rubrum, and the like.

Examples of eukaryotic cells include but are not limited to fungalcells. Examples of fungal cell include, but are not limited to thosebelonging to the genera: Aspergillus, Candida, Chrysosporium,Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora,Penicillium, Pichia, Saccharomyces, and Trichoderma.

Illustrative examples of eukaryotic species include but are not limitedto: Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candidaalbicans, Chrysosporium lucknowense, Fusarium graminearum, Fusariumvenenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta,Pichia finlandica, Pichia kodamae, Pichia membranaefaciens, Pichiamethanolica, Pichia opuntiae, Pichia pastoris, Pichia piperi, Pichiaquercuum, Pichia salictaria, Pichia thermotolerans, Pichia trehalophila,Pichia stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens,Streptomyces aureus, Saccaromyces bayanus, Saccaromyces boulardi,Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomycesgriseochromogenes, Streptomyces griseus, Streptomyces lividans,Streptomyces olivogriseus, Streptomyces rameus, Streptomycestanashiensis, Streptomyces vinaceus, and Trichoderma reesei.

In general, if a eukaryotic cell is used, a non-pathogenic species ispreferred. Illustrative examples of non-pathogenic species include butare not limited to: Fusarium graminearum, Fusarium venenatum, Pichiapastoris, Saccaromyces boulardi, and Saccaromyces cerevisiae.

In addition, certain species have been designated by the Food and DrugAdministration as GRAS or Generally Regarded As Safe. These strainsinclude: Bacillus subtilis, Lactibacillus acidophilus, Lactobacillushelveticus, and Saccharomyces cerevisiae.

IPP Pathways

There are two known biosynthetic pathways that synthesize IPP and itsisomer, dimethylallyl pyrophosphate (“DMAPP”). Eukaryotes other thanplants use the mevalonate-dependent (“MEV”) isoprenoid pathwayexclusively to convert acetyl-coenzyme A (“acetyl-CoA”) to IPP, which issubsequently isomerized to DMAPP. Prokaryotes, with some exceptions, usethe mevalonate-independent or deoxyxylulose 5-phosphate (“DXP”) pathwayto produce IPP and DMAPP separately through a branch point. In general,plants use both the MEV and DXP pathways for IPP synthesis.

MEV Pathway

A schematic representation of the MEV pathway is described in FIG. 1. Ingeneral, the pathway comprises six steps.

In the first step, two molecules of acetyl-coenzyme A are enzymaticallycombined to form acetoacetyl-CoA. An enzyme known to catalyze this stepis, for example, acetyl-CoA thiolase. Illustrative examples ofnucleotide sequences include but are not limited to the followingGenBank accession numbers and the organism from which the sequencesderived: (NC_(—)000913 REGION: 2324131 . . . 2325315; Escherichia coli),(D49362; Paracoccus denitrificans), and (L20428; Saccharomycescerevisiae).

In the second step of the MEV pathway, acetoacetyl-CoA is enzymaticallycondensed with another molecule of acetyl-CoA to form3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to catalyzethis step is, for example, HMG-CoA synthase. Illustrative examples ofnucleotide sequences include but are not limited to: (NC_(—)001145.complement 19061 . . . 20536; Saccharomyces cerevisiae), (X96617;Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907;Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_(—)002758,Locus tag SAV2546, GeneID 1122571; Staphylococcus aureus).

In the third step, HMG-CoA is enzymatically converted to mevalonate. Anenzyme known to catalyze this step is, for example, HMG-CoA reductase.Illustrative examples of nucleotide sequences include but are notlimited to: (NM_(—)206548; Drosophila melanogaster), (NC_(—)002758,Locus tag SAV2545, GeneID 1122570; Staphylococcus aureus),(NM_(—)204485; Gallus gallus), (AB015627; Streptomyces sp. KO 3988),(AF542543; Nicotiana attenuata), (AB037907; Kitasatospora griseola),(AX128213, providing the sequence encoding a truncated HMGR;Saccharomyces cerevisiae), and (NC_(—)001145: complement (115734 . . .118898; Saccharomyces cerevisiae).

In the fourth step, mevalonate is enzymatically phosphorylated to formmevalonate 5-phosphate. An enzyme known to catalyze this step is, forexample, mevalonate kinase. Illustrative examples of nucleotidesequences include but are not limited to: (L77688; Arabidopsisthaliana), and (X55875; Saccharomyces cerevisiae).

In the fifth step, a second phosphate group is enzymatically added tomevalonate 5-phosphate to form mevalonate 5-pyrophosphate. An enzymeknown to catalyze this step is, for example, phosphomevalonate kinase.Illustrative examples of nucleotide sequences include but are notlimited to: (AF429385; Hevea brasiliensis), (NM_(—)006556; Homosapiens), and (NC_(—)001145. complement 712315 . . . 713670;Saccharomyces cerevisiae).

In the sixth step, mevalonate 5-pyrophosphate is enzymatically convertedinto IPP. An enzyme known to catalyze this step is, for example,mevalonate pyrophosphate decarboxylase. Illustrative examples ofnucleotide sequences include but are not limited to: (X97557;Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and(U49260; Homo sapiens).

If IPP is to be converted to DMAPP using the mevalonate pathway, then aseventh step is required. An enzyme known to catalyze this step is, forexample, IPP isomerase. Illustrative examples of nucleotide sequencesinclude but are not limited to: (NC_(—)000913, 3031087 . . . 3031635;Escherichia coli), and (AF082326; Haematococcus pluvialis).

DXP Pathway

A schematic representation of the DXP pathway is described in FIG. 2. Ingeneral, the DXP pathway comprises seven steps. In the first step,pyruvate is condensed with D-glyceraldehyde 3-phosphate to make1-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze this stepis, for example, 1-deoxy-D-xylulose-5-phosphate synthase. Illustrativeexamples of nucleotide sequences include but are not limited to:(AF035440; Escherichia coli), (NC_(—)002947, locus tag PP0527;Pseudomonas putida KT2440), (CP000026, locus tag SPA2301; Salmonellaenterica Paratyphi, see ATCC 9150), (NC_(—)007493, locus tagRSP_(—)0254; Rhodobacter sphaeroides 2.4.1), (NC_(—)005296, locus tagRPA0952; Rhodopseudomonas palustris CGA009), (NC_(—)004556, locus tagPD1293; Xylellafastidiosa Temecula1), and (NC_(—)003076, locus tagAT5G11380; Arabidopsis thaliana).

In the second step, 1-deoxy-D-xylulose-5-phosphate is converted to2C-methyl-D-erythritol-4-phosphate. An enzyme known to catalyze thisstep is, for example, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.Illustrative examples of nucleotide sequences include but are notlimited to: (AB013300; Escherichia coli), (AF148852; Arabidopsisthaliana), (NC_(—)002947, locus tag PP1597; Pseudomonas putida KT2440),(AL939124, locus tag SCO5694; Streptomyces coelicolor A3(2)),(NC_(—)007493, locus tag RSP_(—)2709; Rhodobacter sphaeroides 2.4.1),and (NC_(—)007492, locus tag Pfl_(—)1107; Pseudomonasfluorescens PfO-1).

In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to4-diphosphocytidyl-2C-methyl-D-erythritol. An enzyme known to catalyzethis step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase. Illustrative examples of nucleotide sequences include but arenot limited to: (AF230736; Escherichia coli), (NC_(—)007493, locus tagRSP_(—)2835; Rhodobacter sphaeroides 2.4.1), (NC_(—)003071, locus_tagAT2G02500; Arabidopsis thaliana), and (NC_(—)002947, locus_tag PP1614;Pseudomonas putida KT2440).

In the fourth step, 4-diphosphocytidyl-2C-methyl-D-erythritol isconverted to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. Anenzyme known to catalyze this step is, for example,4-diphosphocytidyl-2C-methyl-D-erythritol kinase. Illustrative examplesof nucleotide sequences include but are not limited to: (AF216300;Escherichia coli) and (NC_(—)007493, locus_tag RSP_(—)1779; Rhodobactersphaeroides 2.4.1).

In the fifth step, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphateis converted to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. An enzymeknown to catalyze this step is, for example, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase. Illustrative examples of nucleotidesequences include but are not limited to: (AF230738; Escherichia coli),(NC_(—)007493, locus_tag RSP_(—)6071; Rhodobacter sphaeroides 2.4.1),and (NC_(—)002947, locus_tag PP1618; Pseudomonas putida KT2440).

In the sixth step, 2C-methyl-D-erythritol 2,4-cyclodiphosphate isconverted to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzymeknown to catalyze this step is, for example,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase. Illustrativeexamples of nucleotide sequences include but are not limited to:(AY033515; Escherichia coli), (NC_(—)002947, locus_tag PP0853;Pseudomonas putida KT2440), and (NC_(—)007493, locus_tag RSP_(—)2982;Rhodobacter sphaeroides 2.4.1).

In the seventh step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate isconverted into either IPP or its isomer, DMAPP. An enzyme known tocatalyze this step is, for example, isopentyl/dimethylallyl diphosphatesynthase. Illustrative examples of nucleotide sequences include but arenot limited to: (AY062212; Escherichia coli) and (NC_(—)002947,locus_tag PP0606; Pseudomonas putida KT2440).

In some embodiments, “cross talk” (or interference) between the hostcell's own metabolic processes and those processes involved with theproduction of IPP as provided by the present invention are minimized oreliminated entirely. For example, cross talk is minimized or eliminatedentirely when the host microorganism relies exclusively on the DXPpathway for synthesizing IPP, and a MEV pathway is introduced to provideadditional IPP. Such a host organisms would not be equipped to alter theexpression of the MEV pathway enzymes or process the intermediatesassociated with the MEV pathway. Organisms that rely exclusively orpredominately on the DXP pathway include, for example, Escherichia coli.

In some embodiments, the host cell produces IPP via the MEV pathway,either exclusively or in combination with the DXP pathway. In otherembodiments, a host's DXP pathway is functionally disabled so that thehost cell produces IPP exclusively through a heterologously introducedMEV pathway. The DXP pathway can be functionally disabled by disablinggene expression or inactivating the function of one or more of the DXPpathway enzymes.

In some embodiments, the host cell produces IPP via the DXP pathway,either exclusively or in combination with the MEV pathway. In otherembodiments, a host's MEV pathway is functionally disabled so that thehost cell produces IPP exclusively through a heterologously introducedDXP pathway. The MEV pathway can be functionally disabled by disablinggene expression or inactivating the function of one or more of the MEVpathway enzymes.

C₁₀ Isoprenoid Compound or Starting Material

In some embodiments GPP is prepared by the method as describedschematically in FIG. 3. One molecule of IPP and one molecule of DMAPPare condensed to form GPP. In some embodiments, the reaction can becatalyzed by an enzyme known to catalyze this step, for example, geranyldiphosphate synthase. Various C₁₀ isoprenoid starting materials can bemade from GPP.

Illustrative examples of nucleotide sequences for geranyl pyrophosphatesynthase include but are not limited to: (AF513111; Abies grandis),(AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686;Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsisthaliana), (AE016877, Locus AP11092; Bacillus cereus; ATCC 14579),(AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ipspini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha xpiperita), (AF182827; Mentha x piperita), (MPI249453; Mentha xpiperita), (PZE431697, Locus CAD24425; Paracoccus zeaxanthinifaciens),(AY866498; Picrorhiza kurrooa), (AY351862; Vitis vinifera), and(AF203881, Locus AAF12843; Zymomonas mobilis).

GPP is then subsequently converted to various C₁₀ isoprenoid startingmaterials using one or more terpene synthases.

Limonene

Limonene, whose structure is

is found in the rind of citrus fruits and peppermint. Limonene is madefrom GPP by limonene synthase. Illustrative examples of suitablenucleotide sequences include but are not limited to: (+)-limonenesynthases (AF514287, REGION: 47 . . . 1867; Citrus limon) and (AY055214,REGION: 48 . . . 1889; Agastache rugosa) and (−)-limonene synthases(DQ195275, REGION: 1 . . . 1905; Picea sitchensis), (AF006193, REGION:73 . . . 1986; Abies grandis), and (MHC4SLSP, REGION: 29 . . . 1828;Mentha spicata).

α-Pinene

α-Pinene having the following structure:

is a constituent of the essential oils from numerous Coniferaceaespecies. Biochemically, α-pinene is made from GPP by a α-pinenesynthase. Some non-limiting examples of suitable nucleotide sequencesinclude GenBank accession numbers AF543530, REGION: 1 . . . 1887((+)-α-pinene) from Pinus taeda and AF543527, REGION: 32 . . . 1921((−)-α-pinene) from Pinus taeda.

β-Pinene

β-Pinene having the following structure:

is a constituent of oil of turpentine. Biochemically, β-pinene is madefrom GPP by a β-pinene synthase. Some non-limiting examples of suitablenucleotide sequences include GenBank accession numbers AF276072, REGION:1 . . . 1749 from Artemisia annua and AF514288, REGION: 26 . . . 1834from Citrus limon.

Sabinene

Sabinene having the following structure:

is a constituent of the essential oil from Juniperus Sabina.Biochemically, sabinene is made from GPP by a sabinene synthase. Anon-limiting example of a suitable nucleotide sequence includes GenBankaccession number AF051901, REGION: 26 . . . 1798 from Salviaofficinalis.

γ-Terpinene

γ-Terpinene, whose structure is

is a constituent of the essential oil from citrus fruits. Biochemically,γ-terpinene is made from GPP by a γ-terpinene synthase. Illustrativeexamples of suitable nucleotide sequences include: (AF514286, REGION: 30. . . 1832 from Citrus limon) and (AB110640, REGION 1 . . . 1803 fromCitrus unshiu).

Terpinolene

Terpinolene, whose structure is

is found in black currant, cypress, guava, lychee, papaya, pine, andtea. Terpinolene is made from GPP by terpinolene synthase. Illustrativeexamples of a suitable nucleotide sequence include but is not limitedto: (AY693650 from Oscimum basilicum) and (AY906866, REGION: 10 . . .1887 from Pseudotsuga menziesii).

In some embodiments, the C₁₀ isoprenoid starting materials can beobtained or prepared from naturally occurred terpenes. Terpenesgenerally include a large and varied class of hydrocarbons, such ashemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes,triterpenes, tetraterpenes, and polyterpenes. Natural terpenes can beproduced by a wide variety of plants, such as Copaifera langsdorfii,conifers, and spurges; insects, such as swallowtail butterflies, leafbeetles, termites, and pine sawflies; and marine organisms, such asalgae, sponges, corals, mollusks and fish.

Copaifera langsdorfii or Copaifera tree is also known as the diesel treeand kerosene tree. It has many names in local languages, includingkupa'y, cabismo, and copaúva. Copaifera tree may produce a large amountof terpene hydrocarbons in its wood and leaves. Generally, one Copaiferatree can produce from about 30 to about 40 liters of terpene oil peryear. The terpene oil can be collected via tapping of the Copaifera treeand subsequently used to formulate or produce various fuel compositions,such as diesel, kerosene, and gasoline, by further processing.

The conifers belong to the plant division Pinophyta or Coniferae and aregenerally cone-bearing seed plants with vascular tissue. The majority ofconifers are trees, but some conifers can be shrubs. Some non-limitingexamples of suitable conifers include cedars, cypresses, douglas-firs,firs, junipers, kauris, larches, pines, redwoods, spruces, and yews.Terpene oils can be obtained from the conifers and subsequently used toformulate or produce various fuel compositions, such as diesel,kerosene, and gasoline, by further processing.

Spurges, also known as Euphorbia, are a very diverse worldwide genus ofplants, belonging to the spurge family (Euphorbiaceae). Consisting ofabout 2160 species, spurges are one of the largest genera in the plantkingdom. The latex or terpene oil of spurges comprises many di- ortri-terpen esters, which can used to formulate or produce various fuelcompositions, such as diesel, kerosene, and gasoline, by furtherprocessing.

In some embodiments, the terpene oil comprises one or more ofhemiterpenes. Hemiterpenes generally comprise a single isoprene unit.Isoprene itself may be considered the only hemiterpene, butoxygen-containing derivatives such as prenol and isovaleric acid arehemiterpenoids.

In certain embodiments, the terpene oil comprises one or more ofmonoterpenes. Monoterpenes generally comprise two isoprene units andhave the molecular formula C₁₀H₁₆. Some non-limiting examples ofmonoterpenes include geraniol and limonene.

In some embodiments, the terpene oil comprises one or more ofsesquiterpenes. Sesquiterpenes generally comprise three isoprene unitsand have the molecular formula C₁₅H₂₄. Some non-limiting examples ofsesquiterpenes include farnesol.

In certain embodiments, the terpene oil comprises one or more ofditerpenes. Diterpenes generally comprise four isoprene units and havethe molecular formula C₂₀H₃₂. They are generally derived fromgeranylgeranyl pyrophosphate. Some non-limiting examples of diterpenesinclude cafestol, kahweol, cembrene and taxadiene (precursor of taxol).

In some embodiments, the terpene oil comprises one or more ofsesterterpenes. Sesterterpenes, generally comprising five isopreneunits, are rare relative to the other terpenes.

In certain embodiments, the terpene oil comprises one or more oftriterpenes. Triterpenes generally comprise six isoprene units and havethe molecular formula C₃₀H₄₈. The linear triterpene squalene, the majorconstituent of shark liver oil, can be derived from the reductivecoupling of two molecules of farnesyl pyrophosphate.

In some embodiments, the terpene oil comprises one or more oftetraterpenes. Tetraterpenes generally comprise eight isoprene units andhave the molecular formula C₄₀H₅₆. Some non-limiting examples oftetraterpenes include the acyclic lycopene, the monocyclicgamma-carotene, and the bicyclic alpha- and beta-carotenes.

In certain embodiments, the terpene oil comprises one or more ofpolyterpenes. Polyterpenes generally comprise two or more isopreneunits. In some embodiments, polyterpenes comprise long chains of manyisoprene units such as natural rubber. Natural rubber generallycomprises polyisoprene in which the double bonds are cis. Some plantsmay produce a polyisoprene with trans double bonds.

Chemical Conversion

Irrespective of its source, each of the C₁₀ isoprenoid startingmaterials and stereoisomers thereof can be chemically converted into afuel component disclosed herein by any known reduction reaction.

The catalyst for the hydrogenation reaction of the isoprenoid startingmaterials or the ring-opening hydrogenation reaction of the bicyclicalkanes disclosed herein can be present in any amount that can cause thereaction to advance by at least about 1%, at least about 2%, at leastabout 3%, at least about 4%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, or at leastabout 30%. The advance of the reaction can be measured as a function ofthe disappearing of one of the reactants or the formation of one of theproducts. For example, an 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30%of advancement in the reaction refers to a decrease in the amount of oneof the reactants by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30%respectively. In some embodiments, the amount of the hydrogenationcatalyst is from about 1 g to about 100 g per liter of reactant, fromabout 2 g to about 75 g per liter of reactant, from about 3 g to about50 g per liter of reactant, from about 4 g to about 40 g per liter ofreactant or from about 5 g to about 30 g per liter of reactant.

The reaction temperature for the hydrogenation reaction of theisoprenoid starting materials or the ring-opening hydrogenation reactionof the bicyclic alkanes disclosed herein can be any temperature that cancause the reaction to advance by at least about 1%, at least about 2%,at least about 3%, at least about 4%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, or atleast about 30%. In some embodiments, the reaction temperature for thehydrogenation reaction of the isoprenoid starting materials is fromabout 10° C. to about 95° C., from about 15° C. to about 85° C., fromabout 20° C. to about 75° C., or from about 20° C. to about 50° C. Inother embodiments, the reaction temperature for the ring-openinghydrogenation reaction of the bicyclic alkanes disclosed herein is fromabout 100° C. to about 500° C., from about 150° C. to about 450° C.,from about 175° C. to about 400° C, or from about 200° C. to about 350°C.

The pressure of the hydrogen for the hydrogenation reaction of theisoprenoid starting materials or the ring-opening hydrogenation reactionof the bicyclic alkanes disclosed herein can be any pressure that cancause the reaction to advance by at least about 1%, at least about 2%,at least about 3%, at least about 4%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, or atleast about 30%. In some embodiments, the pressure of the hydrogen isfrom about 200 psi to about 1000 psi, from about 300 psi to about 800psi, from about 400 psi to about 600 psi, or from about 450 psi to about550 psi.

The reaction time for the hydrogenation reaction of the isoprenoidstarting materials or the ring-opening hydrogenation reaction of thebicyclic alkanes disclosed herein can be any temperature that can causethe reaction to advance by at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, or atleast about 30%. In some embodiments, the reaction time is from about 5minutes to about 24 hours, from about 15 minutes to about 16 hours, fromabout 30 minutes to about 8 hours, or from about 60 minutes to about 4hours.

Optionally, the hydrogen for the hydrogenation reaction of theisoprenoid starting materials or the ring-opening hydrogenation reactionof the bicyclic alkanes disclosed herein can occur in a solvent such asan alkane, a cycloalkane or a combination thereof.

Applications of Fuel Compositions

The fuel composition disclosed herein can be stored in or received by afuel container such as fuel tanks. A fuel tank is generally a safecontainer for flammable liquids. In some embodiments, the fuel tank is apart of a combustion engine system in which a fuel is stored andpropelled by a fuel pump or released in pressurized gas form into acombustion engine. Any fuel tank that can store or receive one or moreliquid fuels can be used herein. Some non-limiting examples of suitablefuel containers include vehicle fuel tanks such as automobile fuel tanksand aircraft fuel tank; fuel tanks above ground or in the ground (e.g.,at a gas station), tanks on transportation vehicles such as tankertrucks, tanker trains, and tanker ships. In certain embodiments, thefuel tank may be connected to other equipments or devices such as powertools, generators and internal combustion engines.

The fuel tanks may vary in size and complexity from small plastic tanksof a butane lighter to the multi-chambered cryogenic Space Shuttleexternal tank. The fuel tank may be made of a plastic such aspolyethylenes (e.g., HDPE and UHDPE) or a metal such as steel oraluminum.

In some embodiments, the fuel composition disclosed here is stored in anaircraft fuel tank and propelled by a fuel pump or released inpressurized gas form into a internal combustion engine to power anaircraft. The aircraft fuel tank can be an integral fuel tank, rigidremovable fuel tank, a bladder fuel tank or a combination thereof.

In certain embodiments, the fuel tank is an integral tank. The integraltank is generally an area inside the aircraft structure that have beensealed to allow fuel storage. An example of this type is the “wetwing”generally used in larger aircraft. Most large transport aircraftgenerally use the integral tank which stores fuel in the wings and/ortail of the airplane.

In some embodiments, the fuel tank is a rigid removable tank. The rigidremovable tank is generally installed in a compartment designed toaccommodate the tank. They generally are made of metal, and may beremoved for inspection, replacement, or repair. The aircraft does notrely on the tank for structural integrity. These tanks are generallyfound in smaller general aviation aircrafts.

In certain embodiments, the fuel tank is a bladder tank. The bladdertank is generally reinforced rubberized bags installed in a section ofaircraft structure designed to accommodate the weight of the fuel. Thebladder tank may be rolled up and installed into the compartment throughthe fuel filler neck or access panel, and may be secured by means ofmetal buttons or snaps inside the compartment. The bladder tank isgenerally found in many high-performance light aircraft and some smallerturboprops.

The fuel composition disclosed herein can be used to power any equipmentsuch as an emergency generator or internal combustion engine, whichrequires a fuel such as jet fuels or missile fuels. An aspect of thepresent invention provides a fuel system for providing an internalcombustion engine with a fuel wherein the fuel system comprises a fueltank containing the fuel composition disclosed herein. Optionally, thefuel system may further comprise an engine cooling system having arecirculating engine coolant, a fuel line connecting the fuel tank withthe internal combustion engine, and/or a fuel filter arranged on thefuel line. Some non-limiting examples of internal combustion enginesinclude reciprocating engines (e.g., gasoline engines and dieselengines), Wankel engines, jet engines, some rocket engines and gasturbine engines.

In some embodiments, the fuel tank is arranged with said cooling systemso as to allow heat transfer from the recirculating engine coolant tothe fuel composition contained in the fuel tank. In other embodiments,the fuel system further comprises a second fuel tank containing a secondfuel for a gasoline engine and a second fuel line connecting the secondfuel tank with the internal combustion engine. Optionally, the first andsecond fuel lines can be provided with electromagnetically operatedvalves that can be opened or closed independently of each other orsimultaneously. In further embodiments, the second fuel is a gasoline.

Another aspect of the invention provides an engine arrangementcomprising an internal combustion engine, a fuel tank containing thefuel composition disclosed herein, a fuel line connecting the fuel tankwith the internal combustion engine. Optionally, the engine arrangementmay further comprise a fuel filter and/or an engine cooling systemcomprising a recirculating engine coolant. In some embodiments, theinternal combustion engine is a gasoline engine. In other embodiments,the internal combustion engine is a jet engine.

When using the fuel composition disclosed herein, it is desirable toremove particulate matter originating from the fuel composition beforeinjecting it into the engine. Therefore, it is desirable to select asuitable fuel filter for use in the fuel system disclosed herein. Waterin fuels used in an internal combustion engine, even in small amounts,can be very harmful to the engine. Therefore, it is desirable that waterpresent in fuel composition can be removed prior to injection into theengine. In some embodiments, water and particulate matter can be removedby the use of a fuel filter utilizing a turbine centrifuge, in whichwater and particulate matter are separated from the fuel composition toan extent allowing injection of the filtrated fuel composition into theengine, without risk of damage to the engine. Other types of fuelfilters that can remove water and/or particulate matter may of coursealso be used.

Another aspect of the invention provides a vehicle comprising aninternal combustion engine, a fuel tank containing the fuel compositiondisclosed herein, a fuel line connecting the fuel tank with the internalcombustion engine. Optionally, the vehicle may further comprise a fuelfilter and/or an engine cooling system comprising a recirculating enginecoolant. Some non-limiting examples of vehicles include cars,motorcycles, trains, ships, and aircraft.

Another aspect of the invention provides a facility for manufacture of afuel, bioengineered fuel component or bioengineered fuel additive of theinvention. In certain embodiments, the facility is capable of biologicalmanufacture of the C₁₀ isoprenoid starting materials. In certainembodiments, the facility is further capable of preparing a substitutedcycloalkane fuel component from the isoprenoid starting material.

The facility can comprise any structure useful for preparing the C₁₀isoprenoid starting material using a microorganism. In some embodiments,the biological facility comprises one or more of the cells disclosedherein. In some embodiments, the biological facility comprises a cellculture comprising at least a C₁₀ isoprenoid starting material in anamount of at least about 1 wt. %, at least about 5 wt. %, at least about10 wt. %, at least about 20 wt. %, or at least about 30 wt. %, based onthe total weight of the cell culture. In further embodiments, thebiological facility comprises a fermentor comprising one or more cellsdescribed herein.

Any fermentor that can provide cells or bacteria a stable and optimalenvironment in which they can grow or reproduce can be used herein. Insome embodiments, the fermentor comprises a culture comprising one ormore of the cells disclosed herein. In other embodiments, the fermentorcomprises a cell culture capable of biologically manufacturing IPP. Infurther embodiments, the fermentor comprises a cell culture capable ofbiologically manufacturing DMAPP. In further embodiments, the fermentorcomprises a cell culture capable of biologically manufacturing GPP fromIPP and DMAPP. In certain embodiments, the fermentor comprises a cellculture comprising at least a C₁₀ isoprenoid starting material in anamount of at least about 1 wt. %, at least about 5 wt. %, at least about10 wt. %, at least about 20 wt. %, or at least about 30 wt. %, based onthe total weight of the cell culture.

The facility can further comprise any structure capable of manufacturingthe fuel component or fuel additive from the C₁₀ isoprenoid startingmaterial. The structure may comprise a hydrogenator for thehydrogenation of the C₁₀ isoprenoid starting materials. Any hydrogenatorthat can be used to reduce a C═C double bond to a C—C single bonds underconditions known to skilled artisans may be used herein. Thehydrogenator may comprise a hydrogenation catalyst disclosed herein. Insome embodiments, the structure further comprises a mixer, a containerand a mixture of the hydrogenation products from the hydrogenation stepand a conventional fuel additive in the container.

Business Methods

One aspect of the present invention relates to a business methodcomprising: (a) obtaining a biofuel comprising at least a substitutedcycloalkane derived from a C₁₀ isoprenoid starting material byperforming a fermentation reaction of a sugar with a recombinant hostcell, wherein the recombinant host cell produces the C₁₀ isoprenoidstarting material; and (b) marketing and/or selling said biofuel.

In other embodiments, the invention provides a method for marketing ordistributing the biofuel disclosed herein to marketers, purveyors,and/or users of a fuel, which method comprises advertising and/oroffering for sale the biofuel disclosed herein. In further embodiments,the biofuel disclosed herein may have improved physical or marketingcharacteristics relative to the natural fuel or ethanol-containingbiofuel counterpart.

In certain embodiments, the invention provides a method for partneringor collaborating with or licensing an established petroleum oil refinerto blend the biofuel disclosed herein into petroleum-based fuels such asa gasoline, jet fuel, kerosene, diesel fuel or a combination thereof. Inanother embodiment, the invention provides a method for partnering orcollaborating with or licensing an established petroleum oil refiner toprocess (for example, hydrogenate, hydrocrack, crack, further purify)the biofuels disclosed herein, thereby modifying them in such a way asto confer properties beneficial to the biofuels. The establishedpetroleum oil refiner can use the biofuel disclosed herein as afeedstock for further chemical modification, the end product of whichcould be used as a fuel or a blending component of a fuel composition.

In further embodiments, the invention provides a method for partneringor collaborating with or licensing a producer of sugar from a renewableresource (for example, corn, sugar cane, bagass, or lignocellulosicmaterial) to utilize such renewable sugar sources for the production ofthe biofuels disclosed herein. In some embodiments, corn and sugar cane,the traditional sources of sugar, can be used. In other embodiments,inexpensive lignocellulosic material (agricultural waste, corn stover,or biomass crops such as switchgrass and pampas grass) can be used as asource of sugar. Sugar derived from such inexpensive sources can be fedinto the production of the biofuel disclosed herein, in accordance withthe methods of the present invention.

In certain embodiments, the invention provides a method for partneringor collaborating with or licensing a chemical producer that producesand/or uses sugar from a renewable resource (for example, corn, sugarcane, bagass, or lignocellulosic material) to utilize sugar obtainedfrom a renewable resource for the production of the biofuel disclosedherein.

EXAMPLES

The following examples are intended for illustrative purposes only anddo not limit in any way the scope of the present invention.

Example 1

This example describes methods for making expression plasmids thatencode enzymes of the MEV pathway from Saccharomyces cerevisiaeorganized in operons, namely the MevT66, MevB, MBI, and MBIS operons.

Expression plasmid pAM36-MevT66, comprising the MevT66 operon, wasgenerated by inserting the MevT66 operon into the pAM36 vector. TheMevT66 operon encodes the set of MEV pathway enzymes that togethertransform the ubiquitous precursor acetyl-CoA to (R)-mevalonate, namelyacetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.Vector pAM36 was generated by inserting an oligonucleotide cassettecontaining AscI-SfiI-AsiSI-XhoI-PacI-FsIl-PmeI restriction enzyme sitesinto the pACYC 184 vector (GenBank accession number XO6403), and byremoving the tet resistance gene in pACYC184. The MevT66 operon wassynthetically generated using SEQ ID No:1 as a template. The nucleotidesequence comprises the atoB gene from Escherichia coli (GenBankaccession number NC_(—)000913 REGION: 2324131 . . . 2325315)codon-optimized for expression in Escherichia coli (encodes anacetoacetyl-CoA thiolase), the ERG13 gene from Saccharomyces cerevisiae(GenBank accession number X96617, REGION: 220 . . . 1695)codon-optimized for expression in Escherichia coli (encodes a HMG-CoAsynthase), and a truncated version of the HGM1 gene from Saccharomycescerevisiae (GenBank accession number M22002, REGION: 1777 . . . 3285)codon-optimized for expression in Escherichia coli (encodes a truncatedHMG-CoA reductase). The synthetically generated MevT66 operon wasflanked by a 5′ EcoRI and a 3′ Hind III restriction enzyme site, andcould thus be cloned into compatible restriction enzyme sites of acloning vector such as a standard pUC or pACYC origin vector. The MevT66operon was PCR amplified with flanking SfiI and AsiSI restriction enzymesites, the amplified DNA fragment was digested to completion using SfiIand AsiSI restriction enzymes, the reaction mixture was resolved by gelelectrophoresis, the approximately 4.2 kb DNA fragment was extracted,and the isolated DNA fragment was inserted into the SfiI and AsiSIrestriction enzyme sites of the pAM36 vector, yielding expressionplasmid pAM36-MevT66.

Expression plasmid pAM25, also comprising the MevT66 operon, wasgenerated by inserting the MevT66 operon into the pAM29 vector. VectorpAM29 was created by assembling the p15A origin of replication and kanresistance gene from pZS24-MCS1 (Lutz and Bujard Nucl Acids Res.25:1203-1210 (1997)) with an oligonucleotide-generated lacUV5 promoter.The DNA synthesis construct comprising the MevT66 operon (see above) wasdigested to completion using EcoRI and Hind III restriction enzymes, thereaction mixture was resolved by gel electrophoresis, the 4.2 kb DNAfragment was extracted using a Qiagen gel purification kit (Valencia,Calif.), and the isolated MevT66 operon fragment was inserted into theEcoRI and HindIII restriction enzyme sites of pAM29 , yieldingexpression plasmid pAM25.

Expression plasmid pMevB-Cm, comprising the MevB operon, was generatedby inserting the MevB operon into the pBBR1MCS-1 vector. The MevB operonencodes the set of enzymes that together convert (R)-mevalonate to IPP,namely mevalonate kinase, phosphomevalonate kinase, and mevalonatepyrophosphate carboxylase. The MevB operon was generated by PCRamplifying from Saccharomyces cerevisiae genomic DNA the ERG12 gene(GenBank accession number X55875, REGION: 580 . . . 1911) (encodes amevalonate kinase), the ERG8 gene (GenBank accession number Z49939,REGION: 3363 . . . 4718) (encodes a phosphomevalonate kinase), and theMVD1 gene (GenBank accession number X97557, REGION: 544 . . . 1734)(encodes a mevalonate pyrophosphate carboxylase), and by splicing thegenes together using overlap extensions (SOEing). By choosingappropriate primer sequences, the stop codons of ERG12 and ERG8 werechanged from TAA to TAG during amplification to introduce ribosomebinding sites into the MevB operon. After the addition of 3′ Aoverhangs, the MevB operon was ligated into the TA cloning vector pCR4(Invitrogen, Carlsbad, Calif.). The MevB operon was excised by digestingthe cloning construct to completion using PstI restriction enzyme,resolving the reaction mixture by gel electrophoresis, and extractingthe 4.2 kb DNA fragment. The isolated MevB operon fragment was ligatedinto the PstI restriction enzyme site of vector pBBR1MCS-1 (Kooach etal., Gene 166(1): 175-176 (1995)), yielding expression plasmid pMevB-Cm.

Expression plasmid pMBI, comprising the MBI operon, was generated byinserting the MBI operon into the pBBR1MCS-3 vector. The MBI operonencodes the same enzymes as the MevB operon, as well as an isopentenylpyrophosphatase isomerase that catalyzes the conversion of IPP to DMAPP.The MBI operon was generated by PCR amplifying the idi gene (GenBankaccession number AF119715) from Escherichia coli genomic DNA usingprimers that contained an XmaI restriction enzyme site at their 5′ ends,digesting the amplified DNA fragment to completion using XmaIrestriction enzyme, resolving the reaction mixture by gelelectrophoresis, extracting the 0.5 kb fragment, and ligating theisolated DNA fragment into the XmaI restriction enzyme site ofexpression plasmid pMevB-Cm, thereby placing idi at the 3′ end of theMevB operon and yielding the MBI operon. The MBI operon was subclonedinto the SalI and SacI restriction enzyme sites of vector pBBR1-MCS-3,yielding expression plasmid pMBI.

Expression plasmid pMBIS, comprising the MBIS operon, was generated byinserting the ispA gene into pMBI. The ispA gene encodes a farnesylpyrophosphate synthase that catalyzes the conversion of IPP to DMAPP.The ispA gene (GenBank accession number D00694, REGION: 484 . . . 1383)was PCR amplified from Escherichia coli genomic DNA using a forwardprimer with a SacII restriction enzyme site and a reverse primer with aSacI restriction enzyme site. The amplified PCR product was digested tocompletion with SacII and SacI restriction enzymes, the reaction mixturewas resolved by gel electrophoresis, and the 0.9 kb fragment wasextracted. The isolated DNA fragment was ligated into the SacII and SacIrestriction enzyme sites of pMBI, thereby placing the ispA gene 3′ ofidi and the MevB operon, and yielding expression plasmid pMBIS.

Expression plasmid pAM45, comprising both the MevT66 operon and the MBISoperon, was generated by inserting the MBIS operon into pAM36-MevT66 andby adding lacUV5 promoters in front of each operon. The MBIS operon wasPCR amplified from pMBIS using primers comprising a 5′ XhoI and a 3′PacI restriction enzyme site. The amplified PCR product was digested tocompletion using XhoI and PacI restriction enzymes, the reaction mixturewas resolved by gel electrophoresis, the 5.4 kb DNA fragment wasextracted, and the isolated DNA fragment was ligated into the XhoI andPacI restriction enzyme sites of pAM36-MevT66, yielding plasmid pAM43. Anucleotide sequence encoding the lacUV5 promoter was then synthesizedfrom oligonucleotides, and sub-cloned into the AscI SfiI and AsiSI XhoIrestriction enzyme sites of pAM43, yielding expression plasmid pAM45.

Example 2

This example describes methods for making expression vectors encodingenzymes of the MEV pathway from Staphylococcus aureus.

Expression plasmid pAM41 was derived from expression plasmid pAM25 byreplacing the HGM1 nucleotide sequence with the mvaA gene. The mvaA geneencodes the Staphylococcus aureus HMG-CoA reductase. The mvaA gene(GenBank accession number BA000017, REGION: 2688925 . . . 2687648) wasPCR amplified from Staphyloccoccus aureus subsp. aureus (ATCC 70069)genomic DNA using primers 4-49 mvaA Spel (SEQ ID No: 2) and 4-49 mvaARXbaI (SEQ ID No: 3), and the amplified DNA fragment was digested tocompletion using SpeI restriction enzyme, the reaction mixture wasresolved by gel electrophoresis, and the approximately 1.3 kb DNAfragment was extracted. The HMG1 nucleotide sequence was removed frompAM25 by digesting the plasmid to completion with HindIII restrictionenzyme. The terminal overhangs of the resulting linear DNA fragment wereblunted using T4 DNA polymerase. The DNA fragment was then partiallydigested using SpeI restriction enzyme, the reaction mixture wasresolved by gel electrophoresis, and the 4.8 kb DNA fragment wasextracted. The isolated DNA fragment was ligated with the SpeI-digestedmvaA PCR product, yielding expression plasmid pAM41.

Expression plasmid pAM52 was derived from expression plasmid pAM41 byreplacing the ERG13 nucleotide sequence with the mvaS gene. The mvaSgene encodes the Staphylococcus aureus HMG-CoA synthase. The mvaS gene(GenBank accession number BA000017, REGION: 2689180 . . . 2690346) wasPCR amplified from Staphyloccoccus aureus subsp. aureus (ATCC 70069)genomic DNA using primers HMGS 5′ Sa mvaS-S (SEQ ID No:4) and HMGS 3′ SamvaS-AS (SEQ ID No:5), and the amplified DNA fragment was used as a PCRprimer to replace the HMG1 gene in pAM41 according to the method ofGeiser et al. BioTechniques 31:88-92 (2001), yielding expression plasmidpAM52.

Expression plasmid pAM97 was derived from expression plasmid pAM45 byreplacing the MevT66 operon with the (atoB(opt):mvaA:mvaA) operon ofexpression plasmid pAM52. Expression plasmid pAM45 was digested tocompletion using AsiSI and SfiI restriction enzymes, the reactionmixture was resolved by gel electrophoresis, and the 8.3 kb DNA fragmentlacking the MevT66 operon was extracted. The (atoB(opt):mvaA:mvaA)operon of pAM52 was PCR amplified using primers 19-25 atoB Sfil-S (SEQID No:6) and 19-25 mvaA-AsiSI-AS (SEQ ID No:7), the PCR product wasdigested to completion using SfiI and AsiSI restriction enzymes, thereaction mixture was resolved by gel electrophoresis, and the 3.7 kb DNAfragment was extracted. The isolated DNA fragment was ligated into theAsiSI and SfiI restriction enzyme sites of expression plasmid pAM45,yielding expression plasmid pAM97.

Expression plasmid pAM97-gpps is derived from expression plasmid pAM97by replacing the ispA nucleotide sequence with a nucleotide sequenceencoding a geranyl diphosphate synthase (“gpps”). The nucleotidesequence encoding the geranyl diphosphate synthase is generatedsynthetically, and comprises the coding sequence of the gpps gene ofArabidopsis thaliana (GenBank accession number Y17376, REGION: 52 . . .1320), codon-optimized for expression in Escherichia coli (SEQ ID No:8).The coding sequence is flanked by a leader NotI restriction enzyme siteand a terminal SacI restriction enzyme site, and can be cloned intocompatible restriction enzyme sites of a cloning vector such as astandard pUC or pACYC origin vector. The synthetically generated geranyldiphosphate synthase sequence is isolated by digesting the DNA synthesisconstruct to completion using NotI and SacI restriction enzymes,resolving the reaction mixture by gel electrophoresis, and extractingthe approximately 1.3 kb DNA fragment. Expression plasmid pAM97 isdigested to completion using NotI and SacI restriction enzymes, thereaction mixture is resolved by gel electrophoresis, the approximately11.2 kb DNA fragment is extracted, and the isolated DNA fragment isligated with the DNA fragment comprising the nucleotide sequenceencoding geranyl diphosphate synthase, yielding expression plasmidpAM97-gpps.

Example 3

This example describes the generation of Escherichia coli host strainsfor the production of α-pinene, γ-terpinene, and terpinolene. Hoststrains were created by transforming chemically competent Escherichiacoli DH1 cells with expression plasmids pMevT, pMBIS-gpps, and one ofthe following: pTrc99A-APS, pTrc99A-GTS, and pTrc99A-TS.

Expression plasmids pTrc99A-APS, pTrc99A-GTS, and pTrc99A-TS weregenerated by inserting a nucleotide sequence encoding an α-pinenesynthase (“APS”), a γ-terpinene synthase (“GTS”), or a terpinolenesynthase (“TS”) into the pTrc99A vector. The nucleotide sequence insertwas generated synthetically, using as a template the coding sequence ofthe α-pinene synthase gene of Pinus taeda (GenBank accession numberAF543530 REGION: 1 . . . 1887), the coding sequence of a γ-terpinenesynthase gene of Citrus limon (GenBank accession number AF514286 REGION:30 . . . 1832), or the coding sequence of the terpinolene synthase geneof Ocimum basilicum (GenBank accession number AY693650) or ofPseudotsuga menziesii (GenBank accession number AY906866 REGION: 10 . .. 1887), all nucleotide sequences being codon-optimized for expressionin Escherichia coli. (The codon-optimized nucleotide sequences of theα-pinene, γ-terpinene, and terpinolene synthases are shown as SEQ IDNos:9 through 12). The coding sequences were flanked by a leader XmaIrestriction enzyme site and a terminal XbaI restriction enzyme site. Thesynthetic nucleic acids were cloned into compatible restriction enzymesites of a cloning vector such as a standard pUC or pACYC origin vector,from which they could be liberated again by digesting the DNA synthesisconstruct to completion using XbaI and XmaI restriction enzymes,resolving the reaction mixture by gel electrophoresis, and gelextracting the approximately 1.8 to 1.9 terpene synthase encoding DNAfragment. The isolated DNA fragment was ligated into the XbaI XmaIrestriction enzyme site of vector pTrc99A (Amman et al., Gene 40:183-190(1985)), yielding expression plasmids pTrc99A-APS, pTrc99A-GTS, orpTrc99A-TS (see FIG. 4 for plasmid maps).

Host cell transformants were selected LB agar containing 100 ug/mLcarbenicillin, 34 ug/mL chloramphenicol, and 5 ug/mL tetracycline.Single colonies were transferred from LB agar to culture tubescontaining 5 mL of LB liquid medium and antibiotics as detailed above.The cultures were incubated by shaking at 37° C. until growth reachedlate exponential phase. The cells were stored at −80° C. in cryo-vialsin 1 mL frozen aliquots made up of 400 uL 50% glycerol and 600 uL liquidculture.

Example 4

This example describes the generation of Escherichia coli host strainsfor the production of D-limonene, β-pinene, and sabinine. Host strainsare created by transforming chemically competent Escherichia coli DH1cells with expression plasmids pMevT, pMBIS-gpps, and one of thefollowing: pTrc99A-LMS, pTrc99A-BPS, and pTrc99A-SS.

Expression plasmids pTrc99A-LMS, pTrc99A-BPS, and pTrc99A-SS aregenerated by inserting a nucleotide sequence encoding a D-limonenesynthase (“LMS”), β-pinene synthase (“BPS”), or sabinine synthase (“SS”)into the pTrc99A vector. The nucleotide sequence inserts are generatedsynthetically, using as a template for example the coding sequence ofthe D-limonene synthase gene of Abies grandis (GenBank accession numberAF006193 REGION: 73 . . . 1986), the coding sequence of the β-pinenesynthase of Artemisia annua (GenBank accession number AF276072 REGION: 1. . . 1749), or the coding sequence of the sabinine synthase gene ofSalvia officinalis (GenBank accession number AF051901 REGION: 26 . . .1798). The nucleotide sequences encoding the β-pinene and sabininesynthases are flanked by a leader XmaI restriction enzyme site and aterminal XbaI restriction enzyme site, and the nucleotide sequenceencoding the D-limonene synthase is flanked by a leader NcoI restrictionenzyme site and a terminal PstI restriction enzyme site. The DNAsynthesis constructs are digested to completion using XmaI and XbaI (forthe β-pinene and sabinine synthase constructs), or NcoI and PstIrestriction enzymes (for the D-limonene synthase construct). Thereaction mixtures are resolved by gel electrophoresis, the approximately1.7 to 1.9 kb DNA fragments are gel extracted, and the isolated DNAfragments are ligated into the XmaI XbaI restriction enzyme site (forthe β-pinene and sabinine synthase inserts), or the NcoI PstIrestriction enzyme site (for the D-limonene synthase insert) of thepTrc99A vector, yielding expression plasmids pTrc99A-LMS, pTrc99A-BPS,and pTrc99A-SS (see FIG. 4 for plasmid maps).

Host cell transformants are selected LB agar containing 100 ug/mLcarbenicillin, 34 ug/mL chloramphenicol, and 5 ug/mL tetracycline.Single colonies are transferred from LB agar to culture tubes containing5 mL of LB liquid medium and antibiotics as detailed above. The culturesare incubated by shaking at 37° C. until growth reaches late exponentialphase. The cells are stored at −80° C. in cryo-vials in 1 mL frozenaliquots made up of 400 uL 50% glycerol and 600 uL liquid culture.

Example 5

This example describes the production of α-pinene, γ-terpinene,terpinolene, D-limonene, β-pinene, and sabinine in an Escherichia colihost strain of Examples 4 or 5.

Seed cultures are grown overnight by adding the 1 mL stock aliquot to a125 mL flask containing 25 mL M9-MOPS-0.5% Yeast Extract, 2% glucose,100 ug/mL carbenicillin, 34 ug/mL chloramphenicol, and 5 ug/mLtetracycline. The cultures are used to inoculate 250 mL baffled flaskscontaining 40 mL M9-MOPS-0.5% Yeast Extract, 2% glucose, and antibioticsas detailed above at an initial OD₆₀₀ of approximately 0.05. Culturesare incubated by shaking at 30° C. on a rotary shaker at 250 rpm untilthey reach an OD₆₀₀ of 0.2, at which point the production of thecompound of interest in the host cells is induced with 1 mM IPTG (40 uLof 1M IPTG added to the culture medium). The compound of interest isseparated from the culture medium through solvent-solvent extraction, orby settling and decantation if the titer of the compound of interest islarge enough to saturate the media and to form a second phase.

Example 6

This example describes the generation of Saccharomyces cerevisiae hoststrains for the production of α-pinene, γ-terpinene, terpinolene,D-limonene, β-pinene, and sabinine. Host strains are generated by firstgenerating a Saccharomyces cerevisiae strain that produces elevatedlevels of geranyl pyrophosphate, and then transforming the strain withexpression plasmid pRS425-APS, pR425-BPS, pR425-GTS, pR425-TS,pR425-LMS, pR425-BPS, or pR425-SS.

The generation of host strain EPY219 is described in Ro et al. (Nature440: 940-943; 2006) and in PCT Patent Publication WO2007/005604. Hoststrain EPY219 is cured of expression plasmid pRS425ADS by growth in YPDmedium (Methods in Yeast Genetics: A Cold Spring Harbor LaboratoryCourse Manual, 2005 ed., ISBN 0-87969-728-8), plating for singlecolonies on YPD agar, and then patching single colonies onto CSM-Met-Hisagar and CSM-Met-HisLeu agar. Clones that grow on CSM-Met-His agar butnot on CSM-Met-His-Leu agar are cured (i.e., have lost the plasmidpRS425ADS). One such clone is then transformed with plasmid pδ-gpps.Plasmid pδ-gpps is generated by inserting a synthetically generatednucleotide sequence comprising the coding sequence of the gpps gene ofArabidopsis thaliana (GenBank accession number Y17376, REGION: 52 . . .1320) into vector pRS-Sacll-DX (Ro et al. Nature 440: 940-943; 2006),digesting the resulting plasmid using SacII restriction enzyme, gelextracting the expression cassette fragment, and cloning the isolatedfragment into the SacII restriction enzyme site of pδ-UB. Transformantsare initially selected on SD-URA-HIS-MET plates, and then cultured andplated on SD-HIS-MET plates including 1 g L⁻¹ 5-FOA for the constructionof a Saccharomyces cerevisiae strain that produces elevated levels ofgeranyl pyrophosphate.

Expression plasmids pRS425-APS, pR425-GTS, pR425-TS, pR425-BPS,pR425-LMS, pR425-BPS, and pR425-SS are generated by inserting anucleotide sequence encoding an α-pinene synthase (“APS”), γ-terpinenesynthase (“GTS”), terpinolene synthase (“TS”), D-limonene synthase(“LMS”), β-pinene synthase (“BPS”), or sabinine synthase (“SS”) into thepRS425-Gal1 vector (Mumberg et. al. (1994) Nucl. Acids. Res. 22(25):5767-5768). The nucleotide sequence insert is generated synthetically,using as a template for example the coding sequence of the α-pinenesynthase gene of Pinus taeda (GenBank accession number AF543530 REGION:1 . . . 1887), the coding sequence of a γ-terpinene synthase gene ofCitrus limon (GenBank accession number AF514286 REGION: 30 . . . 1832),the coding sequence of the terpinolene synthase gene of Ocimum basilicum(GenBank accession number AY693650) or of Pseudotsuga menziesii (GenBankaccession number AY906866 REGION: 10 . . . 1887), the coding sequence ofthe D-limonene synthase gene of Abies grandis (GenBank accession numberAF006193 REGION: 73 . . . 1986), the coding sequence of the β-pinenesynthase of Artemisia annua (GenBank accession number AF276072 REGION: 1. . . 1749), or the coding sequence of the sabinine synthase gene ofSalvia officinalis (GenBank accession number AF051901 REGION: 26 . . .1798). The synthetically generated nucleotide sequence is flanked by aleader BamHI site and a terminal XhoI site, and can thus be cloned intocompatible restriction enzyme sites of a cloning vector such as astandard pUC or pACYC origin vector. The synthetically generatednucleotide sequence is isolated by digesting the DNA synthesis constructusing BamHI and XhoI restriction enzymes (partial digest for theα-pinene and γ-terpinene synthase constructs, complete digests for allother constructs), the reaction mixture is resolved by gelelectrophoresis, the approximately 1.7 to 1.9 kb DNA fragment comprisingthe terpene synthase coding sequence is gel extracted, and the isolatedDNA fragment is ligated into the BamHI XhoI restriction enzyme site ofthe pRS425-Gal1 vector, yielding expression plasmid pRS425-APS,pR425-GTS, pR425-TS, pR425-BPS, pR425-LMS, pR425-BPS, or pR425-SS.

Host cell transformants are selected on synthetic defined media,containing 2% glucose and all amino acids except leucine (SM-glu).Single colonies are transferred to culture vials containing 5 mL ofliquid SM-glu lacking leucine. The cultures are incubated by shaking at30° C. until growth reaches stationary phase. The cells are stored at−80° C. in cryo-vials in 1 mL frozen aliquots made up of 400 μL 50%glycerol and 600 μL liquid culture.

Example 7

This example describes the production of α-pinene, γ-terpinene,terpinolene, D-limonene, β-pinene, and sabinine in a Saccharomycescerevisiae host strain of Example 6.

Seed flasks are grown overnight by adding the 1 mL stock aliquot to 25mL of SM-glu lacking leucine in a 125 mL flask. The cultures are used toinoculate 250 mL baffled flasks containing 40 mL of synthetic definedmedia lacking leucine, 0.2% glucose, and 1.8% galactose at an initialOD₆₀₀ of approximately 0.05. The cultures are incubated by shaking at30° C. on a rotary shaker at 200 rpm. Because the presence of glucose inthe media prevents induction of the Gal1 promoter by galactose,production of the compound of interest is not induced until the cellsuse up the glucose in the media and switch to using galactose as theirmain carbon source. The compound of interest is separated from the mediathrough solvent-solvent extraction, or by settling and decantation ifthe titer of the compound of interest is large enough to saturate themedia and form a second phase.

Example 8

A design of experiments (DOE) methodology was used to test sixcatalysts. Three factors were tested: catalyst type, temperature, andcatalyst loading. The catalysts used for the hydrogenation screen were:5% rhodium on activated carbon from Alfa Aesar stock #11761, 5%ruthenium on carbon from Alfa Aesar stock #L00524, 5% platinum on carbonfrom Alfa Aesar stock #L00566, platinum oxide from Sigma-Aldrich stock#206032, 5% palladium on carbon dry from Alfa Aesar stock #A12623, andnickel on silica-alumina from Alfa Aesar stock #31276. This DOE resultedin 12 experiments, to which were added 3 center points using Platinum oncarbon. The catalyst loadings were normalized to mmol/L of the metal,which took into account the different molecular weights and percentagesof metal per gram of catalyst. The catalyst loading was varied from 1.3mmol/L to 6.5 mmol/L, while the temperature was varied from 200° C. to300° C.

All hydrogenation experiments were carried out in 75 mL Parr pressurereactors with 20 mL starting material (48.869% cis-pinane, 50.137%trans-pinane). Experiments were conducted in the following manner:starting material was added to the reactor, followed by the specifiedamount of catalyst. The reactor was then charged with 200 psi ofhydrogen, stirred, and heated to the desired temperature. At this point,the reactor was re-charged with 600 psi of hydrogen. All reactions weremonitored by computer and re-charged to 600 psi as needed, allowing thereactions to drop at least 100 psi before re-charging. Each experimentwas run for four days, or until it stopped taking up hydrogen for atleast 6 hours. The products were analyzed by GC/MS and mass spectra werecompared against the NIST database for identification. Chromatogramswere then integrated to get product distribution. No furtherpurification was attempted.

Of the six catalysts tested, rhodium on carbon and ruthenium on carbonshowed strong selectivity towards tetramethylcyclohexane at 200° C. Dueto the positive result of the ruthenium on carbon catalyst, two moreexperiments were attempted, one at 150° C., and one at 250° C., as wellas a repeat of the initial experiment. The reaction showed activity atboth temperatures. However the experiment at 150° C. did not go tocompletion over the course of two days, although all of the cis-pinanewas consumed leaving only trans-pinane. Some of the higher temperatureruns, at 250° C. and above, showed multiple unidentified products, twoof which were later identified as trimethylcyclohexane andethyl-methylcyclohexane. At 300° C., palladium, platinum, and rutheniumon carbon yielded both isomers of p- and o-cymene, as well. Overall, thehighest selectivity and conversion to product came from the tworuthenium on carbon experiments, one of which yielded 91.9% totaltetramethylcyclohexanes, and the other yielded 92.7%. Rhodium on carbonyielded 86.3% total tetramethylcyclohexane, however it was unable toconvert a significant amount of starting material, and also producedmore p- and o-menthanes than other experiments. Taking the average ofthe two ruthenium runs at 200° C., one obtains a product ratio ofapproximately 32:1 in favor of tetramethylcyclohexane over all otherknown products, as compared to 21:1 for the best rhodium run. Theconversion for the Ruthenium processes at 200° C. was approximately99.5%. The results of the DOE experiment are shown in Table 1.

In Table 1, Comps. 1 & 2 refer to tetramethylcyclohexane compounds (1)and (2); Comps. 14 & 17 refer to menthane compounds (14) and (17); Comp.11 refers to aromatic compound (11); Comp. 27 refers to dimethyloctane(27); and Comp. 24 refers to pinane compound (24).

TABLE 1 Cat. Products^(#) (wt. %) Temp Loading Comps. Comps. Expt.Catalyst (° C.) (mmol/L) 1 & 2 14 & 17 Comp. 11 Comp. 27 Comp. 24 1 Pd/C300 1.3 31.635 26.606 0.551 16.098 15.688 2 Pd/C 200 6.5 54.248 4.017 00 35.659 3 Ru/C 300 1.3 41.348 16.171 5.023 14.841 9.473 4 Ru/C 200 6.591.894 1.151 0 0.117 0.496 5 PtO2 200 1.3 0.123 0 0 0 99.085 6 PtO2 3006.5 3.404 11.678 0 24.495 54.116 7 Ni/SiAl 200 1.3 4.476 0.646 0 088.258 8 Ni/SiAl 300 6.5 33.798 21.24 0 6.38 18.724 9 Rh/C 200 1.386.335 3.55 0 0.074 7.053 10 Rh/C 300 6.5 38.405 18.185 0 13.384 28.6211 Pt/C 300 1.3 8.831 4.934 17.159 19.173 38.207 12 Pt/C 200 6.5 0.2810.023 0 0.045 98.581 13 Pt/C 250 3.9 0.159 0.398 0 1.641 96.861 14 Pt/C250 3.9 0.361 0.491 0 2.128 95.903 15 Pt/C 250 3.9 0.348 0.235 0 1.05997.25 16 Ru/C 200 6.5 92.685 3.064 0 0.2 0.416 17 Ru/C 250 6.5 91.782 00 0.254 0.624 18 Ru/C 150 6.5 41.295 4.085 0 0 51.939

Example 9

A composition designated as AMG-500 was made by hydrogenating pinane totetramethylcyclohexane using 5% ruthenium on carbon at 200° C. Theseconditions resulted in a 92% product yield. The product composition ofAM-500 was determined to be: i) 52% cis-1,1,2,3-tetramethylcyclohexane;ii) 8% trans-1,1,2,3-tetramethylcyclohexane; iii) 32%1,1,2,5-tetramethylcyclohexane; iv) 3% menthane; v) and 0.4%trans-pinane. The 1,1,2,5-tetramethylcyclohexane had two isomers; one at28% and the other at 4% but could not determine which was the cis ortrans isomer. Similarly, the methane had two isomers; one at 2% and theother at 1% but could not determine which was the p-menthane or theo-menthane.

AMG-500 and blends of AMG-500 in CARBOB were tested in various ASTMtests and the results are summarized in Tables 2 and 3 below.

TABLE 2 ASTM AMG-500/CARBOB Blend Test Unleaded Gasoline CARBOB (vol. %AMG-500 in CARBOB) AMG-500 Property Method Units ASTM D4814Specification 0 5 20 50 100 Ocatane Number, Research (RON) D2699 — 88.488.7 89.3 90.7 94.7 Ocatane Number, Motor (MON) D2700 — 81.6 81.9 82.584.7 87.9 Anti Knock Index, (R + M)/2 — 85.0 85.3 85.9 87.7 91.3 Calc.Blending RON — 94.4 92.9 93.0 Calc. Blending MON — 87.6 86.1 87.8 Calc.Blending Anti Knock Index — 91.0 89.5 90.4 Vapor Pressure (CARBEquation) D5191 psi max.  7.8-15.0* 5.70 5.47 4.77 3.26 0.06 VaporPressure (EPA Equation) D5191 psi max.  7.8-15.0* 5.81 5.57 4.89 3.400.22 Vapor Pressure (ASTM Equation) D5191 psi max.  7.8-15.0* 5.95 5.725.04 3.56 0.41 Calc. Blending Vapor Pressure (CARB) psi 1.1 1.1 0.8Calc. Blending Vapor Pressure (EPA) psi 1.0 1.2 1.0 Calc. Blending VaporPressure (ASTM) psi 1.4 1.4 1.2 Distillation Initial Boiling Point(Evaporated) D86 ° F. max. 122-158* 107 109 112 119 313 10% (Evaporated)D86 ° F. min. 150-170* 146 148 154 184 316 50% (Evaporated) D86 ° F.max. 230-250* 215 222 247 299 321 90% (Evaporated) D86 ° F. max.365-374* 322 325 328 328 323 Final Boiling Point (Evaporated) D86 ° F.max. 437 389 389 378 376 360 Residue D86 vol. % max. 2.0 1.3 1.1 1.4 0.90.8 Vapor-Liquid Ratio Temperature (V/L = 20) D5188 ° F. min.  95-140*162.0 165.9 >176.0 >176.0 >176.0 Driveability Index max. 1200-1250* 11851213 1299 1502 1760

TABLE 3 ASTM Unleaded Gasoline AMG-500/CARBOB Blend Test ASTM CARBOB(vol. % AMG-500 in CARBOB) AMG-500 Property Method Units D4814Specification 0 5 20 50 100 Density @ D4052 g/cm³ 0.7421 0.7456 0.75220.7740 0.8044 0 15.0° C. (59.0° F.) Heat of D4809 BTU/lb 20,026 19,81919,964 20,110 19,906 Combustion, Gross Energy Content BTU/gal 124,027123,324 125,326 129,901 133,634 (HHV) Copper Corrosion D130 max. No. 11a 1a 1a 1a 1a Silver Corrosion D4814A max. 1 0 0 0 0 0 OxidationStability Induction D525 min. 240 >240 >240 >240 >240 >240 Period @100.0° C. Total Potential D873 mg/100 mL max. 6.0 1.3 1.3 1.4 1.5 1.0Residue Gum Content, D381 mg/100 mL 0.5 0.5 1.0 1.0 2.0 Unwashed GumContent, D381 mg/100 mL max. 5.0 0.0 0.0 0.0 0.5 1.0 Washed Sulfur D5453ppm max. 30 12 11 8.8 4.9 <1 Phosphorous D3231 g P/gal max. 0.005<0.0008 <0.0008 <0.0008 0.0008 0.0013 Lead (Pb) D3237 g Pb/gal max. 0.05<0.02 <0.02 <0.02 <0.02 <0.02 Benzene D5580 vol. % max. 1.0 0.61 0.530.50 0.32 <0.01 Total Aromatics D5580 vol. % max. 25 24.26 22.91 19.4412.33 0.06 Total Olefins D6550 vol. % max. 6.0 5.9/5.0 5.4/4.7 4.3/3.72.8/2.4 <1 ***** Add 10% ethanol and run tests below Water Tolerance,D6422 ° C. max. −41-10* <−24 <−24 <−24 <−24 <−24 Phase Separation DryVapor D5191 psi max.  7.8-15.0* 7.01 6.60 5.98 4.66 1.93 PressureEquivalent (CARB) Calc. Blending psi −1.2 1.9 2.3 Vapor Pressure (CARB)

FIGS. 5A and 5B show the distillation profile of AMG-500 and blends ofAMG-500 in CARBOB respectively.

The fuel composition disclosed herein can be produced in acost-effective and environmentally friendly manner. Advantageously, theisoprenoid compounds provided herein can be produced by one or moremicroorganisms. These isoprenoid compounds can thus provide a renewablesource of energy as a substitute for petroleum-based fuel such asgasoline. Further, these isoprenoid compounds can decrease dependence onnon-renewable sources of fuel, fuel components and/or fuel additives. Incertain embodiments, the present invention encompasses a fuelcomposition comprising a bioengineered tetramethylcyclohexane.

As demonstrated above, embodiments of the invention provide various fuelcompositions which are particularly useful as jet fuels or missilefuels. While the invention has been described with respect to a limitednumber of embodiments, the specific features of one embodiment shouldnot be attributed to other embodiments of the invention. No singleembodiment is representative of all aspects of the invention. In someembodiments, the compositions or methods may include numerous compoundsor steps not mentioned herein. In other embodiments, the compositions ormethods do not include, or are substantially free of, any compounds orsteps not enumerated herein. Variations and modifications from thedescribed embodiments exist. For example, the jet fuel compositionsdisclosed herein need not comprising at least a tetramethylcyclohexane.It can comprise any type of hydrocarbons generally suitable for jet fuelapplications. It should be noted that the application of the fuelcompositions disclosed herein is not limited to gasoline engines; theycan be used in any equipment which requires gasoline. Although there arespecifications for most gasoline compositions, not all gasolinecompositions disclosed herein need to meet all requirements in thespecifications. It is noted that the methods for making and using thefuel compositions disclosed herein are described with reference to anumber of steps. These steps can be practiced in any sequence. One ormore steps may be omitted or combined but still achieve substantiallythe same results. The appended claims intend to cover all suchvariations and modifications as falling within the scope of theinvention.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A fuel composition comprising: (a) a tetramethylcyclohexane in anamount of at least 5 wt. %, based on the total weight of the fuelcomposition; and (b) a fuel component.
 2. The fuel composition of claim1, wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 3. The fuel composition of claim1, wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 4. The fuel composition of claim1, wherein the fuel component is a petroleum-based fuel component. 5.The fuel composition of claim 4, wherein the petroleum-based fuelcomponent is gasoline.
 6. The fuel composition of claim 4, wherein thepetroleum-based fuel component is jet fuel.
 7. The fuel composition ofclaim 4, wherein the petroleum-based fuel component is kerosene.
 8. Thefuel composition of claim 1, wherein the fuel component is a coal-basedfuel component.
 9. The fuel composition of claim 1 further comprising afuel additive.
 10. The fuel composition of claim 9, the fuel additive isselected from the group consisting of oxygenates, antioxidants, thermalstability improvers, cetane improvers, stabilizers, cold flow improvers,combustion improvers, anti-foams, anti-haze additives, corrosioninhibitors, lubricity improvers, icing inhibitors, injector cleanlinessadditives, smoke suppressants, drag reducing additives, metaldeactivators, dispersants, detergents, demulsifiers, dyes, markers,static dissipaters, biocides and combinations thereof.
 11. The fuelcomposition of claim 1, wherein the amount of the tetramethylcyclohexaneis at most about 30 wt. %, based on the total weight of the fuelcomposition.
 12. The fuel composition of claim 1, wherein the amount ofthe tetramethylcyclohexane is at most about 20 wt. %, based on the totalweight of the fuel composition.
 13. The fuel composition of claim 1,wherein the amount of the tetramethylcyclohexane is at most about 10 wt.%, based on the total weight of the fuel composition.
 14. The fuelcomposition of claim 1 comprising:

or at least one stereoisomer thereof;

or at least one stereoisomer thereof;

or at least one stereoisomer thereof; and

or at least one stereoisomer thereof, wherein the total amount of (a)and (b) is from about 1 wt. % to about 99 wt. %, and the total amount of(c) and (d) is from 0.5 wt. % to about 50 wt. %, based on the totalweight of (a)-(d).
 15. The fuel composition of claim 14 furthercomprising:

or at least one stereoisomer thereof;

and

or at least one stereoisomer thereof; wherein the amount of (e) is from0 wt. % to about 50 wt. %, the amount of (f) is from about 0.1 wt. % toabout 20 wt. %, and the amount of (g) is from about 0.1 wt. % to about30 wt. %, based on the total weight of (a)-(g).
 16. The fuel compositionof claim 15, wherein the total amount of (a) and (b) is from about 50wt. % to about 99 wt. %, based on the total weight of (a)-(g).
 17. Thefuel composition of claim 15, wherein the total amount of (a) and (b) isfrom about 80 wt. % to about 99 wt. %, based on the total weight of(a)-(g).
 18. The fuel composition of claim 15, wherein the total amountof (c) and (d) is less than about 10 wt. %, based on the total weight of(a)-(g).
 19. The fuel composition of claim 1 that is an RBOB, CARBOB orAZ RBOB.
 20. The fuel composition of claim 1 that has a Reid vaporpressure between 7.0 psi and 15.0 psi.
 21. A method of making a fuelcomposition comprising contacting pinene with hydrogen in the presenceof a hydrogenation catalyst to form at least a tetramethylcyclohexane.22. The method of claim 21, wherein the pinene is α-pinene, β-pinene ora combination thereof.
 23. The method of claim 21, wherein thehydrogenation catalyst comprises a ruthenium catalyst.
 24. The method ofclaim 23, wherein the ruthenium catalyst is ruthenium on a supportmaterial.
 25. The method of claim 24, wherein the support material iscarbon.
 26. The method of claim 21, further comprising the step ofmixing the tetramethylcyclohexane with a fuel component to make the fuelcomposition.
 27. The method of claim 26, wherein the fuel component is apetroleum-based fuel component.
 28. The method of claim 27, wherein thepetroleum-based fuel component is gasoline.
 29. The method of claim 27,wherein the petroleum-based fuel component is jet fuel.
 30. The methodof claim 27, wherein the petroleum-based fuel component is kerosene. 31.A vehicle comprising an internal combustion engine; a fuel tankconnected to the internal combustion engine; and a fuel composition inthe fuel tank, wherein the fuel composition comprises at least 5 wt. %of a tetramethylcyclohexane, based on the total weight of the fuelcomposition, and a fuel component, and wherein the fuel composition isused to power the internal combustion engine.
 32. The vehicle of claim31, wherein the internal combustion engine is a gasoline engine.
 33. Thevehicle of claim 31, wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 34. The vehicle of claim 31,wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 35. A vehicle comprising aninternal combustion engine; a fuel tank connected to the internalcombustion engine; and a fuel composition in the fuel tank, wherein thefuel composition is prepared by contacting pinene with hydrogen in thepresence of a hydrogenation catalyst, and wherein the fuel compositionis used to power the internal combustion engine.
 36. The method of claim35, wherein the pinene is α-pinene, β-pinene or a combination thereof.37. The vehicle of claim 35, wherein the internal combustion engine is agasoline engine.
 38. A method of making a fuel composition comprising:(a) contacting a cell capable of making pinene with a sugar underconditions suitable for making pinene; (b) converting the pinene topinane; (c) converting the pinane to at least a tetramethylcyclohexane;and (d) mixing the tetramethylcyclohexane with a fuel component to makethe fuel composition.
 39. The method of claim 38, wherein the pinene isconverted to pinane by hydrogen in the presence of a first hydrogenationcatalyst.
 40. The method of claim 38, wherein the pinane is converted totetramethylcyclohexane by hydrogen in the presence of a secondhydrogenation catalyst.
 41. A method of making an RBOB comprising mixinga gasoline with a fuel composition comprising a tetramethylcyclohexanehaving a quaternary carbon atom in the cyclohexane ring, wherein theRBOB has a Reid vapor pressure from about 7.0 psi to about 15.0 psi, andwherein the amount of the amount of the tetramethylcyclohexane is fromabout 1 wt. % to about 50 wt. %, based on the total weight of the fuelcomposition.
 42. The method of claim 41, wherein thetetramethylcyclohexane is

or at least one stereoisomer thereof.
 43. The method of claim 41,wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 44. A fuel tank containing a fuelcomposition, wherein the fuel composition comprises at least 5 wt. % ofa tetramethylcyclohexane, based on the total weight of the fuelcomposition, and a fuel component.
 45. The fuel tank of claim 44,wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 46. The fuel tank of claim 44,wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 47. The fuel tank of claim 44,wherein the fuel tank is a vehicle fuel tank.
 48. A fuel compositioncomprising a gasoline and at least one tetramethylcyclohexane having aquaternary carbon atom in the cyclohexane ring, wherein the amount ofthe tetramethylcyclohexane is from about 1 wt. % to about 50 wt. %,based on the total weight of the fuel composition.
 49. The fuelcomposition of claim 48, wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 50. The fuel composition of claim48, wherein the tetramethylcyclohexane is

or at least one stereoisomer thereof.
 51. The fuel composition of claim50 further comprising a fuel additive.
 52. The fuel composition of claim51, the fuel additive is selected from the group consisting ofoxygenates, antioxidants, thermal stability improvers, cetane improvers,stabilizers, cold flow improvers, combustion improvers, anti-foams,anti-haze additives, corrosion inhibitors, lubricity improvers, icinginhibitors, injector cleanliness additives, smoke suppressants, dragreducing additives, metal deactivators, dispersants, detergents,demulsifiers, dyes, markers, static dissipaters, biocides andcombinations thereof.